Solution and solid-phase formation of glycosidic linkages

ABSTRACT

The invention relates to methods that permit the rapid construction of oligosaccharides and other glycoconjugates. Methods for forming multiple glycosidic linkages in solution in a single step are disclosed. The invention takes advantage of the discovery that the relative reactivity of glycoside residues containing anomeric sulfoxides and nucleophilic functional groups can be controlled. In another aspect of the invention, the reactivity of activated anomeric sugar sulfoxides is utilized in a solid phase method for the formation of glycosidic linkages. The methods disclosed may be applied to the preparation of specific oligosaccharides and other glycoconjugates, as well as to the preparation of glycosidic libraries comprising mixtures of various oligosaccharides, including glycoconjugates, which can be screened for biological activity.

Aspects of the present invention were supported by HHS R01 GM42733,HHS/NRSA F32 GM15051, and ONRN00014-91-J-1230. The United StatesGovernment has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part (CIP) of priorco-pending U.S. application Ser. No. 08/198,271, filed Feb. 18, 1994,which is in turn a CIP of prior co-pending U.S. application Ser. No.08/021,391, filed Feb. 23, 1993, the complete disclosures of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to methods that permit the rapidconstruction of oligosaccharides and other glycoconjugates. Moreparticularly, the present invention relates to methods for formingmultiple glycosidic linkages in solution in a single step. The presentinvention takes advantage of the discovery that the relative reactivityof glycoside residues containing anomeric sulfoxides and nucleophilicfunctional groups can be controlled. In another aspect of the presentinvention, the reactivity of activated anomeric sugar sulfoxides isutilized in a solid phase method for the formation of glycosidiclinkages. The methods disclosed may be applied to the preparation ofspecific oligosaccharides and other glycoconjugates, as well as to thepreparation of glycosidic libraries comprising mixtures of variousoligosaccharides, including glycoconjugates, which can be screened forbiological activity.

BACKGROUND OF THE INVENTION 2.1. General Background

The oligosaccharide chains of glycoproteins and glycolipids playimportant roles in a wide variety of biochemical processes. Found bothat cell surfaces and circulating in biological fluids, these glycosidicresidues act as recognition signals that mediate key events in normalcellular development and function. They are involved in fertilization,embryogenesis, neuronal development, hormonal activities, inflammation,cellular proliferation, and the organization of different celltypes intospecific tissues. They are also involved in intracellular sorting andsecretion of glycoproteins as well as in the clearance of plasmaglycoproteins from circulation.

In addition to their positive role in the maintenance of health,oligosaccharides are also involved in the onset of disease. Forinstance, oligosaccharides on cell surfaces function as receptors forviruses and toxins, as well as more benign ligands. Modified cellsurface carbohydrates have been implicated in tumorigenesis andmetastasis. The oligosaccharide structures that mediate inflammation andhelp prevent infection can, when produced at excessive levels, stimulatethe development of chronic inflammatory disease. (Some references on theroles of oligosaccharides produced by eukaryotes in health and diseaseinclude: Hakomori TIBS, 1984, 45; Feizi et al. TIBS, 1985, 24;Rademacher et al. Ann. Rev. Biochem. 1988, 57, 785; Feizi TIBS, 1991,84; Dennis and Laferte Cancer Res. 1985, 45, 6034; Fishman J. Membr.Biol. 1982, 69, 85; Markwell et al. PNAS USA, 1981, 78, 5406; Wiley andSkehel J. Annu. Rev. Biochem. 1987, 56, 365; Kleinman et al. PNAS USA,1979, 76, 3367; Walz et al. Science 1990, 250.)

Although bacteria do not produce the same types of oligosaccharides orother glycoconjugates as eukaryotes, procaryotes nevertheless produce awide variety of glycosylated molecules. Many such molecules have beenisolated and found to have antitumor or antibiotic activity. Bacteriallyproduced glycosylated molecules having potential therapeutic utilityinclude chromomycin, calicheamicin, esperamicin, and the ciclamycins. Inall these cases, the carbohydrates residues have been shown to beimportant to biological activity. However, the precise functions of thecarbohydrate residues are not well understood and there is nounderstanding of structure-activity relationships.

Because of their diverse roles in health and disease, oligosaccharideshave become a major focus of research. It is widely accepted that thedevelopment of technology to 1) detect and 2) block or otherwiseregulate some of the abnormal functions of oligosaccharides would leadto significant improvements in health and well-being. Moreover, itshould be possible to exploit some of the normal functions ofoligosaccharides (e.g., various recognition processes) for otherpurposes, including drug delivery to specific cell types. In addition,it may be possible to develop new antitumor agents from syntheticglycosylated molecules reminiscent of glycosylated bacterial antitumoragents.

There are ongoing efforts to develop products related tooligosaccharides, including diagnostic kits for detecting carbohydratesassociated with various diseases, vaccines to block infection by virusesthat recognize cell surface carbohydrates, drug delivery vehicles thatrecognize carbohydrate receptors, and monoclonal antibodies, whichrecognize abnormal carbohydrates, for use as drugs. The timelydevelopment of these and other carbohydrate-based biomedical productsdepends in turn on the availability of technologyto produceoligosaccharides and other glycoconjugates rapidly, efficiently, and inpractical quantities for basic and developmental research.

In particular, there is a need for methods that permit the rapidpreparation of glycosidic libraries comprising mixtures of variousoligosaccharides or other glycoconjugates which could then be screenedfor a particular biological activity. It has been shown, for example,that screening of mixtures of peptides is an efficient way ofidentifying active compounds and elucidating structure-activityrelationships. There are numerous ways to generate chemically diversemixtures of peptides and determine active compounds. See, for example,Furka et al,, Int. J. Peptide Protein Res. 1992, 27, 487; Lam et al.Nature 1991, 354, 82; Houghten Nature 1991, 354, 84; Zuckermann et al.Proc. Natl. Acad. Sci. USA 1992, 89, 4505; Petithory Proc. Natl. Acad.Sci. USA, 1991, 88, 11510; Geyse Proc. Natl. Acad. Sci. USA, 1984, 82,3998; Houghten Proc. Natl. Acad. Sci. USA, 1985, 82, 5131; Fodor Science1991, 251, 767. We are not aware of effective methods to generatediverse mixtures of oligosaccharides and other glycoconjugates forscreening purposes.

2.2. Anthracyclines

Ciclamycin 0 (1, below), an anthracycline antibiotic isolated fromStreptomyces capoamus, possesses high inhibitory in vitro activityagainst experimental tumors. This drug is comprised of the aglyconeε-pyrromycinone and a trisaccharide. See, Bieber et al. J. Antibiot.1987, 40, 1335. The trisaccharide contains two repeating units of2-deoxy-L-fucose (A, B) and one unit of the keto sugar (C),L-cinerulose. All the sugars are connected to each other through a 1-4axial linkage.

Although ciclamycin was discovered almost thirty years ago, little isunderstood about its function because insufficient quantities areavailable from natural sources. Consequently, the best way to obtainciclamycin in large quantites, and the only way to obtain its analogs,is through chemical synthesis. ##STR1##

The aglycone of ciclamycin, ε-pyrromycinone, can be obtained bydeglycosylation of other readily available antibiotics, such asmarcellomycin, musettamycin and cinerubin. Efficient strategies exist inthe literature for coupling the trisaccharide to the aglycone. See, forexample, Kolar et al. Carbohydr. Res. trisaccharide suffer fromlimitations of overall ease and efficiency.

Anthracycline antibiotics occur as intermediates in the metabolism ofseveral Streptomyces species. They are potent chemotherapeutic drugsthat have been used extensively in the treatment of various solid tumorsand leukemias. See, Arcamone, F. Doxorubicin Anticancer Antibiotics;Academic Press: New York, 1981. The aglycone of all anthracyclinesconsists of a tricyclic quininoid system with a functionalizedcyclohexane moiety. Various substitution patterns frequently encounteredamong the aglycones are outlined, below. ##STR2##

A common feature of all anthracycline antibiotics is an oligosaccharideresidue attached to the C-7 hydroxyl group of the aglycone. The sugarresidue at this position can be a mono, di or trisaccharide. The mostfrequently encountered sugars include daunosamine, rhodosamine,2-deoxy-L-fucose and L-cinerulose. ##STR3##

On the basis of several studies conducted on the anthracyclineantibiotics daunomycin, adriamycin, and aclacinomycin, it has becomeincreasingly clear that the oligosaccharide components of these naturalDNA binders play an important role in DNA binding and recognition. See,Bieber et al, supra. However, little is known about the actual functionof the sugars, in part because it is difficult to selectively 0 modifythese drugs. The first chemical synthesis of ciclamycin was accomplishedby S. J. Danishefsky and coworkers. See, Suzuki et al. J. Am. Chem. Soc.1990, 112, 8895.

2.2.1. Synthesis of 2-Deoxy Oligosaccharides

Complex glycoconjugates like anthracyclines and aureolic acids are ofconsiderable scientific and pharmaceutical interest and have beenapplied extensively in cancer chemotherapy. A common structural featurein these compounds is the presence of 2-deoxy oligosaccharides. Indeed,several types of alpha- and beta-2-deoxy glycosides are frequently foundin naturally occurring bioactive molecules. In addition to the aureolicacid antibiotics and anthracycline antibiotics, there can be foundcardiac glycosides, avermectins, erythromycins, and the enediyneantibiotics. The efficient construction of these 2-deoxy glycosides,particularly 2-deoxy-β-glycosides, has been a long-standing problem incarbohydrate chemistry. Controlling the β stereoselectivity in 2-deoxysugars is difficult because there can be no stereo-directing anchimericassistance from the C-2 position.

In general, the specific therapeutic effect of these drugs is thought tobe caused by the aglycone, while the sugars are thought to beresponsible for regulating the pharmacokinetics. It is hoped that bymodifying the carbohydrate moiety, it is possible to increase theefficacy and also decrease the cytotoxicity of these drugs.

The development of sugar analogs requires good synthetic methods for theconstruction of 2-deoxy oligosaccharides. Unfortunately, glycosylationmethods available for synthesis of 2-deoxy oligosaccharides aregenerally unsatisfactory. Since 2-deoxy glycosyl donors lack asubstituent at the C-2 position, they are unstable. They decomposerapidly in most glycosylation reactions, thereby resulting in pooryields of glycosides.

In fact, one of the better existing methods for constructing 2-deoxyoligosaccharides, the glycal method, circumvents this problem by notactually using 2-deoxyglycosyl donors directly. This procedure, which isone of the most widely used glycosylation methods for constructing2-deoxy glycosides, involves a two-step process. In the first step, a1,2-anhydro sugar (glycal) is treated with a suitable electrophile, E⁺,to form a 1,2-onium intermediate. Nucleophilic attack from the oppositeside affords the glycoside, with 1,2-trans-configured bonds. In thesecond step, the substituent at C-2 is removed to form the desired2-deoxy glycoside.

2.3. Solution Methods for Obtaining Oligosaccharides

There are currently two general ways to obtain oligosaccharides. Thefirst is by isolation from natural sources. This approach is limited tonaturally occurring oligosaccharides that are produced in largequantities. The second way is through enzymatic or chemical synthesis.The variety of oligosaccharides available through enzymatic synthesis islimited because the enzymes used can only accept certain substrates.Chemical synthesis is more flexible than enzymatic synthesis and has thepotential to produce an enormous variety of oligosaccharides. Theproblem with chemical synthesis has been that it is extremely expensivein terms of time and labor. This problem is a consequence of the way inwhich the chemical synthesis of oligosaccharides has been carried out todate.

Oligosaccharides are formed from monosaccharides connected by glycosidiclinkages. In a typical chemical synthesis of an oligosaccharide, a fullyprotected glycosyl donor is activated and allowed to react with aglycosyl acceptor (typically another monosaccharide having anunprotected hydroxyl group) in solution. The glycosylation reactionitself can take anywhere from a few minutes to days, depending on themethod used. The coupled product is then purified and chemicallymodified to transform it into a glycosyl donor. The chemicalmodification may involve several steps, each single step requiring asubsequent purification. (A "single step" is defined as a chemicaltransformation or set of transformations carried out in a "single"reaction vessel without the need for intermediate isolation orpurification steps.) Each purification is time consuming and can resultin significant losses of material. The new glycosyl donor, adisaccharide, is then coupled to another glycosyl acceptor. The productis then isolated and chemically modified as before. It is not unusualfor the synthesis of a trisaccharide to require ten or more steps fromthe component monosaccharides. In one recent example, the fullyprotected trisaccharide side chain of an antitumor antibiotic calledciclamycin 0 was synthesized in 14 steps with a 9% yield based on thecomponent monosaccharides. See, Suzuki et al, supra. Thus, the time andexpense involved in the synthesis of oligosaccharides has been a seriousobstacle to the development of carbohydrate drugs and other biomedicalproducts.

One way to increase the speed and efficiency of oligosaccharidesynthesis is to develop methods that permit the construction of multipleglycosidic linkages in a single step. Before the present discovery, theapplicants are unaware of a one-step method which involves theregioselective formation of multiple glycosidic bonds and which providesa rapid, efficient and high yield process for the production ofoligosaccharides.

2.4. Solid-Phase Synthesis of Oligosaccharides

Besides reducing the number of steps involved in the synthesis ofoligosaccharides, one can also increase the speed and efficiency of asynthetic process by eliminating the need for isolation andpurification. Theoretically, elimination of the need for isolation andpurification could be achieved by developing a solid-phase process forthe synthesize of oligosaccharides.

Due to the magnitude of the potential advantages of solid-phasesynthesis, there have been previous attempts to synthesizeoligosaccharides on a solid phase. Solid-phase methods for synthesismake isolation and purification unnecessary because excess reagents anddecomposition products can simply be washed away from the resin-boundproduct. This advantage translates into an enormous savings in terms oftime, labor, and yield. (The advantages of solid-phase methods oversolution methods for the synthesis of peptides and nucleic acids havebeen amply demonstrated. These advantages would, of course, extend to asolid-phase synthesis of oligosaccharides. For the solid-phase synthesisof peptides, see, for example, Barany, G. and Merrifield, R. B. 1980, inThe Peptides, Gross, E. and Meienhofer, J. Eds., Academic Press, NewYork, Vol 2, pp. 1-284.)

As far back as 1971, Frechet and Schuerch outlined the requirements forsolid-phase oligosaccharide synthesis. See, Frechet and Schuerch J. Am.Chem. Soc. 1971, 93, 492. First, the resin must be compatible with thereaction conditions. Second, the solid support must contain appropriatefunctionality to provide a link to the glycosidic center (or elsewhere),which link is inert to the reaction conditions but can be easily cleavedto remove the oligosaccharide upon completion of the synthesis. Third,appropriate protecting group schemes must be worked out so thatparticular hydroxyls can be selectively unmasked for the next couplingreaction. The other hydroxyls should be protected by "permanent"blocking groups to be removed at the end of the synthesis. Fourth, theglycosylation reactions should be efficient, mild, and go to completionto avoid failure sequences. Fifth, the stereochemistry of the anomericcenters must be maintained during the coupling cycles and should bepredictable based on the results obtained in solution for any givendonor/acceptor pair. Sixth, cleavage of the permanent blocking groupsand the link to the polymer must leave the oligosaccharide intact.

Unfortunately, although it has been generally accepted that solid-phaseoligosaccharide synthesis is a desirable goal, and although Frechet andSchuerch (as well as others) were able to outline a strategy forsolid-phase oligosaccharide synthesis, no one, before the presentdiscovery, had been able to implement such a strategy. In previousattempts to synthesize oligosaccharides on insoluble resins, thecoupling yields were low and the stereochemical control was inadequate,particularly for the construction of β-glycosidic linkages (i.e.,1,2-trans glycosidic linkages in which the glycosidic bond at theanomeric position of the sugar is trans to the bond bearing the sugarsubstituent at C-2).

These problems have been attributed to the fact that reaction kineticson the solid phase are slower than they are in solution. See, Eby andSchuerch, Carbohydr. Res. 1975, 39, 151. The consequence of suchunfavorable kinetics is that most glycosylation reactions, which maywork reasonably well in solution, simply do not work well on a solidphase both in terms of stereochemical control and yield. Thus, forexample, Frechet and Schuerch found that two glycosylation reactions,which both involve the displacement of an anomeric halide in thepresence of a catalyst, gave predominantly the β-anomer (i.e., the1,2-trans product) in solution but gave mixtures on the solid phase.Frechet and Schuerch concluded that it would be necessary to useneighboring group participation to form β-glycosidic linkages on thesolid phase.

Again, however, it has been found that neighboring participating groups(NPGs) frequently deactivate glycosyl donors to the point that existingglycosylation methods could not be adapted to the solid phase.Frequently, glycosyl donors would decompose in the resin mixture beforeglycosylation can take place. See, Eby and Schuerch, supra. In someinstances the resin has also been known to decompose due to theharshness of the conditions required for glycosylation. Furthermore, formany ester-type NPGs, there is a significant problem with acyl transferfrom the glycosyl donors to the free glycosyl acceptors on the resin.This side reaction caps the resin and prevents further reaction.

Frechet has reviewed the problems encountered in trying to implement astrategy for solid-phase oligosaccharide synthesis. See, Frechet,Polymer-supported Reactions in Organic Synthesis, p. 407, P. Hodge andD. C. Sherrington, Eds., John Wiley & Sons, 1980. He has concluded thatsolid-phase oligosaccharide synthesis is not yet competitive withsolution synthesis "due mainly to the lack of suitable glycosylationreactions."

There have been some efforts to overcome the unfavorable reactionkinetics associated with solid-phase reactions by using soluble resins.In the best example to date Douglas et al. used a soluble polyethyleneglycol resin with a succinic acid linker and achieved 85-95% couplingyields using a glycosylation method known for over 80 years (theKoenigs-Knorr reaction) with excellent control of anomericstereochemistry. See, Douglas et al. J. Am. Chem. Soc. 1991, 113, 5095.Soluble resins may have advantages for some glycosylation reactionsbecause they offer a more "solution-like" environment. However,step-wise synthesis on soluble polymers requires that the intermediatebe precipitated after each step and crystallized before another sugarresidue can be coupled.

Moreover, several additions of the same glycosylating reagent aretypically required to push a reaction to completion. In the above case,for example, Douglas et al. had to repeat the same coupling reactionfive times to achieve a high yield. Each repetition requires aprecipitation step to wash the reagents away. Product may be lost witheach precipitation step. In addition, repeated precipitations make theprocess very time-consuming. Thus, the soluble resin approach tooligosaccharide synthesis fails to provide all the potential advantagesassociated with solid phase synthesis using insoluble resins.

A new method for glycosylation involving anomeric sugar sulfoxides wasreported by Kahne and co-workers. See, Kahne et al. J. Am. Chem. Soc.1989, 111, 6881. The anomeric sugar sulfoxides were activated withequimolar amounts of triflic anhydride in the presence of a hinderedbase. The triflic anhydride-activated glycosyl donors proved to be quitereactive in solution and could be used to glycosylate extremelyunreactive substrates under mild conditions. However, this report waslimited to solution reactions, and there was no suggestion thatsolid-phase reactions could be carried out with any degree of utility.

Thus, the state of the art underscores the prevailing and unfullfilledneed for a glycosylation method which provides for the rapid, efficient,and high yield synthesis of oligosaccharides. Moreover, an efficientsynthesis of oligosaccharides on the solid phase has not beendemonstrated which provides all the previously mentioned advantages ofsolid-phase methods.

SUMMARY OF THE INVENTION

The present invention provides methods for constructing multipleglycosidic linkages in solution using anomeric sugar sulfoxides asglycosyl donors and for constructing sequential glycosidic linkages onthe solid phase, with control over the stereochemical configuration ofthe anomeric bond. Thus, depending upon the selected conditions andstarting materials, α- or β-anomers can be produced on the solid phaseusing anomeric sugar sulfoxides as glycosyl donors. The methods of thepresent invention may be applied to the preparation of specificoligosaccharides or glycoconjugates or to the preparation of mixtures ofvarious oligosaccharides or glycoconjugates for the creation ofglycosidic libraries that can subsequently be screened to detectcompounds having a desired biological activity.

The present invention also relates to the discovery that the activationof anomeric sulfoxides with catalytic quantities of an activating agentprovides very good yields of condensation product under very mildconditions. Preferably, the activating agent is a strong organic acid,such as trifluoromethanesulfonic or "triflic" acid (TfOH),p-toluenesulfonic acid (TsOH) or methanesulfonic acid (MsOH), mostpreferably, TfOH. In particular, it has been found that for theconstruction of 2-deoxyglycosides, the catalytic glycosylation proceduredescribed herein is considered the method of choice. A preferredembodiment of this aspect of the invention, involving the synthesis of2-deoxy glycosides via the triflic acid-catalyzed glycosylation, isdescribed in greater detail, below.

Other objects of the present invention will be apparent to one ofordinary skill on consideration of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a method of synthesizing the protected trisaccharideof ciclamycin 0 in one step from the component monosaccharides.

FIG. 2 illustrates a process of forming homopolymers of 2-deoxy fucosein one step.

FIGS. 3A and 3B illustrate a process of synthesizing mixtures ofglycoconjugates having biological activity, including potential DNAbinding activity. The glycoconjugates so produced can subsequently bescreened (e.g., for DNA binding activity) to evaluate the preferredlength and the preferred sugar residues of the oligosaccharide portionof the glycoconjugate based on the activity being tested.

FIGS. 4A and 4B illustrate methods of forming and removing two exemplarytypes of linkages from a solid support (e.g., polystyrene resin).

FIGS. 5A and 5B illustrate an apparatus used to carry out solid phaseoligosaccharide synthesis.

FIG. 6 illustrates the general scheme for synthesis of a β-linkeddisaccharide on the solid phase.

FIG. 7 illustrates the general scheme for synthesis of an α-linkeddisaccharide on the solid phase.

FIG. 8 illustrates the general scheme for synthesis of a trisaccharideon the solid phase.

FIGS. 9A and 9B are a ¹ H NMR spectrum of monosaccharide 1 of FIG. 1.

FIGS. 10A and 10B are a ¹ H NMR spectrum of monosaccharide 2 of FIG. 1.

FIGS. 11A and 11B are a ¹ H NMR spectrum of monosaccharide 3 of FIG. 1.

FIGS. 12A and 12B are a ¹ H NMR spectrum of trisaccharide 5 of FIG. 1.

FIGS. 13A and 13B present an expanded region of the ¹ H NMR spectrum of5, in which the anomeric protons of the trisaccharide are labeled.

FIGS. 14A and 14B are a ¹ H NMR spectrum of disaccharide 4 of FIG. 1.

FIG. 15 represents a scheme for the synthesis of ciclamycin 0.

FIGS. 16A and 16B represent a scheme for the synthesis of atrisaccharide.

FIGS. 17A and 17B represent a scheme for the synthesis of selecteddisaccharides.

FIG. 18 provides a schematic representation of the solid phase synthesisof (1-3)alpha- and (1-3)beta-linked polysaccharides in a stereospecificfashion starting with glucosamine derivative, 11.

DETAILED DESCRIPTION OF THE INVENTION

What follows is a detailed description of the preferred embodiments ofthe present invention.

5.1. Definitions

Activating agent: A chemical agent that on addition to a glycosylsulfoxide reacts with the anomeric sulfoxide group, thus rendering theanomeric carbon susceptible to nucleophilic attack. In the case ofbifunctional sugars or glycosidic residues, the activating agent is alsoable to deprotect a blocked nucleophilic group under the same conditionsused to activate the anomeric sulfoxide group.

Acid scavenger: A chemical agent such as any base that sequestersprotons, thereby minimizing side reactions that are promoted by acidicconditions.

Sulfenic acid scavenger: A chemical agent such as methyl propiolate thatspecifically sequesters sulfenic acid, typically resulting in theformation of an unreactive monophenyl sulfoxide. In the absence of asulfenic acid scavenger, sulfenic acid reacts with itself to formdiphenyl disulfide monosulfoxide and water. Water interferes with theglycosylation reaction.

Covalent linker moiety: A covalent linker moiety is any chemical groupthat allows the covalent attachment of the glycosyl acceptor to theinsoluble solid support. The covalent linker moiety may comprisealiphatic or aromatic groups and may contain functional groups such asethers, amides, esters or combinations of same.

Bifunctional: The characteristic of a sugar or glycosidic residue to beable to function on activation both as a glycosyl donor and a glycosylacceptor under the conditions of the single-step process of the presentinvention.

Biological activity: Any activity displayed by a compound or moleculewhich has potential physiologic, pharmacologic, diagnostic, ortherapeutic applications.

Carbohydrate receptor: Any molecule that binds any carbohydrate.Typically the molecule is a macromolecule such as a protein or DNA.

Glycoconjugate: Any compound or molecule that is covalently bound to aglycosidic residue.

Glycoside: Any sugar containing at least one pentose or hexose residuein which the anomeric carbon bears a non-hydrogen substituent.Typically, the non-hydrogen substituent is a heteroatom, such asnitrogen, oxygen, phosphorus, silicon or sulfur.

Glycosyl acceptor: Any compound that contains at least one nucleophilicgroup which, under the conditions of the single-step process of thepresent invention, is able to form a covalent bond with the anomericcarbon of a glycosyl donor. As referred to herein, a glycosyl acceptoris any sugar or glycoconjugate that contains unprotected hydroxyl,amino, or mercapto groups or such groups that are blocked by protectinggroups that can be removed in situ, i.e., under the conditions of thesingle-step process of the present invention.

Glycosyl donor: A sugar or glycosidic residue that bears a sulfoxidegroup at the anomeric carbon, which group on activation renders theanomeric carbon susceptible to attack by the nucleophilic group of aglycosyl acceptor to form the glycosidic linkage.

Glycosidic libraries: A mixture of oligosaccharides of varying sequenceswhich can be subjected to a screening procedure to identify compounds ormolecules that exhibit biological activity. Such libraries may alsoinclude various glycoconjugates.

Masked group: A masked group is any chemical group that is stable underthe conditions of the solution or solid phase glycosylation reactiondescribed herein but which can be converted, when desired, into an aminogroup or a derivative thereof, such as an acetylated or sulfated aminogroup.

Monofunctional glycosyl acceptor: A glycosyl acceptor as in thedefinition above, with the additional provision that the capacity to actas a glycosyl donor at the same time (i.e., under the conditions of thesingle step process of the present invention) is specifically excluded.

Monofunctional glycosyl donor: A glycosyl donor as in the definitionabove, with the additional provision that the capacity to act as aglycosyl acceptor at the same time (i.e., under the conditions of thesingle step process of the present invention) is specifically excluded.

Monofunctional glycosyl unit: A sugar that is either a glycosyl acceptoror a glycosyl donor but does not have the capacity to function as bothupon activation under the conditions of the single step process of thepresent invention.

Oligosaccharides: A glycosidic residue having three or moremonosaccharide units joined by glycosidic linkages.

Potential glycosyl acceptor: Any compound containing at least onenucleophilic group which is potentially able to form a covalent bondwith the anomeric carbon of a glycosyl donor.

Resin: Any insoluble solid support to which the glycosyl acceptor may becovalently attached. Synthetic polymers or copolymers, such aspolystyrene or crosslinked styrene/divinyl-benzene copolymer, which areinsoluble in organic solvents used in the sulfoxide mediatedglycosylation reaction, are suitable for use in the present invention.

Single step reaction: A single step reaction is defined as a chemicaltransformation or set of transformations carried out in a "single"reaction vessel without the need for intermediate isolation orpurification steps (i.e., a one-step or one-pot reaction).

Temporal protecting group: A blocking or protecting group that can beremoved in situ, preferably, but not necessarily, under the sameconditions used to activate an anomeric sulfoxide group.

5.2. General Methods

The following general methods have been divided into two maincategories: the first concerns solution reactions involving theformation of multiple glycosidic bonds and the second relates to thesynthesis of oligosaccharides in which the growing oligomer is bound toa solid support.

5.3. Formation Of Multiple Glycosidic Linkages In Solution

One or more glycosyl donors having alkyl or aryl sulfoxides at theanomeric position and one or more glycosyl acceptors having one or morefree hydroxyls and/or other nucleophilic groups (e.g., amines) and/orsilyl ether protected hydroxyls are combined in a reaction vessel. Theresulting mixture may include both monofunctional glycosyl donors andglycosyl acceptors as well as bifunctional glycosyl units, i.e.,saccharides that can function simultaneously as glycosyl donors andacceptors. However, in order to form more than one glycosidic linkage(i.e., to produce a trisaccharide or longer product), at least one ofthe reactants must be a bifunctional glycosyl unit.

The glycosyl acceptors and donors may be blocked by a suitableprotecting group, including, but not limited to, ether, ester,acetamido, or thioester protecting groups, at one or more positions.However, it is understood that an ester (or acetamido or thioester)protecting group at C-2 of a glycosyl donor will influence thestereochemical outcome of glycosylation, resulting in a 1,2-transglycosidic bond.

The mixture of glycosyl donors and acceptors is dissolved underanhydrous conditions in a non-nucleophilic solvent, including, but notlimited to, toluene, ether, tetrahydrofuran (THF), methylene chloride,chloroform, propionitrile, or mixtures thereof. It has been found thatthe choice of solvent influences the stereochemical outcome ofglycosylation for reactions in which neighboring group participation isnot involved. In general, for a given donor/acceptor pair, the use of anon-polar solvent, such as toluene, results in the formation of a higherpercentage of alpha isomer, while the use of a more polar solvent, suchas propionitrile, results in formation of a higher percentage of thebeta anomer.

The reaction is initiated by the addition of an effective amount of anactivating agent. In a particular embodiment of the present invention,0.5 equiv. of triflic anhydride, plus 1.5 equiv. base (as an acidscavenger), are added to the reaction mixture. (Equivalents are relativeto glycosyl sulfoxide.) A catalytic amount of triflic acid (e.g., <0.05equiv.) can also be used, preferably along with excess sulfenic acidscavenger (e.g., ca. 20 equiv. of methyl propiolate). It has been foundthat catalytic triflic acid is preferred when the reaction mixturecontains 2-deoxy glycosyl donors or when one of the glycosyl acceptorsin the reaction is a silyl ether. On the other hand, triflic anhydrideis preferred when maximum reactivity of the glycosyl donors isimportant. However, it should be noted that the moderately basicconditions that obtain with the use of triflic anhydride are noteffective to deprotect certain silyl ethers (e.g., t-butylsilyl ethers).Moreover, although the use of triflic anhydride plus2,6-ditert-butyl-4-methylpyridine will result in the in situdeprotection of trimethylsilyl ethers, the use of triflic anhydride plusa stronger base (such as Hunig's base) will not. Thus, both activatingagents can be used in reactions involving a bifunctional glycosyl unitcontaining a silyl ether protected hydroxyl, although triflic anhydrideonly works under a specific set of conditions (choice of base, choice ofsilyl protecting group). Otherwise, the two activation methods areusually interchangeable.

The methyl propiolate or other sulfenic acid scavenger and/or activatedmolecular sieves may be added to the reaction either before or justafter the addition of activating agent. Sulfenic acid scavengerssignificantly improve the yield of glycosylation when catalytic triflicacid is used as the activating agent.

The reaction is normally carried out at low temperature (preferably inthe range of about -78° C. to as low as bout -100° C.) but may beallowed to proceed at higher temperatures, in some cases as warm as roomtemperature.

The reaction is quenched by the addition of aqueous bicarbonate andextracted. The reaction mixture may then be subjected to a purificationprocedure and/or the product(s) deprotected if necessary. The proceduremay be used to construct specific oligosaccharides or mixtures ofvarious oligosaccharides or other glycoconjugates for screening forbiological activity.

In particular embodiments of the present invention, it has beendiscovered that the reactivity of different glycosyl donors may bemodulated by manipulating the chemical structure and electronic natureof the anomeric sulfoxide. Such manipulation is due, in part, to thefinding that the rate-limiting step in the glycosylation reaction isactivation of the sulfoxide to the action of the activating agent. Itwas subsequently shown that the reactivity of the glycosyl sulfoxidescan be influenced by manipulating the nucleophilicity of the sulfoxideoxygen.

Generally, the more nucleophilic the sulfoxide oxygen, the faster theglycosylation reaction. Thus, electron-donating substituents on the R'group attached to the sulfoxide increase the nucleophilicity of thesulfoxide oxygen and speed up the rate of the reaction. By contrast,electron-withdrawing groups decrease the nucleophilicity of thesulfoxide oxygen and slow down the reaction. For example, perbenzylatedglucosyl p-methoxyphenyl sulfoxide reacts faster than the correspondingunsubstituted phenyl sulfoxide, while perbenzylated glucosylp-nitrophenyl sulfoxide reacts slower than the correspondingunsubsituted phenyl sulfoxide.

The ability to influence the nucleophilicity of different sulfoxides andhence to manipulate the reactivity of different glycosyl donors has beenexploited in particular embodiments of the present invention. Forexample, this ability permits sequential glycosylations to take place insolution, as illustrated in FIG. 1.

In yet other embodiments of the present invention, multiple glycosidiclinkages are formed in solution using silylated glycosyl acceptors.Silyl ethers are excellent glocosyl acceptors when catalytic triflicacid is the activating agent and trimethylsilyl ethers work well asglycosyl acceptors when triflic anhydride is the activating agent and2,6-ditert-butyl-4-methylpyridine is the base. However, they must beunmasked in order to couple. (Hence, there is a requirement for slightlyacidic conditions in the glycosylation reaction when silylethers areused as glycosyl acceptors.) Because silyl ethers must be unmasked inorder to couple, they react more slowly than unprotected alcohols. Inthis manner, it has been demonstrated that one can modulate thereactivity of two otherwise similar glycosyl acceptors by selective useof silyl protecting groups.

In selected embodiments of the present invention, the distribution ofthe length of the oligosaccharides or the glycosidic residues of theglycoconjugates produced can be influenced by varying the ratio ofmonofunctional glycosyl acceptors and monofunctional glycosyl donors tobifunctional glycosyl units in the reaction mixture. For example, it hasbeen shown that higher ratios of monofunctional glycosyl acceptors tobifunctional glycosyl units in the reaction mixture lead to shorterlength polymers. The total concentration of reactants also influencesthe length distribution. (See, Sections 6.6 and 6.8 and FIGS. 2 and 3,below.)

In particular embodiments of the present invention, it may be desirableto include only two or three different types of sugars in the reactionmixture and to manipulate the reactivity of the glycosyl donors andacceptors so that a specific oligosaccharide is produced. An example ofthis procedure is given in Sections 6.1. and 6.6, below.

Yet in other embodiments of the present invention, it may be desirableto include several different types of sugars in the reaction mixture inorder to generate a chemically diverse mixture of oligosaccharides orglycoconjugate products for the creation of libraries that may bescreened for biological activity. An example of such a method isillustrated in Section 6.6 and FIG. 3, below.

The chemical diversity can be influenced by manipulation of the numberof different sugars included in the mixture. The chemical diversity willalso be a function of the order in which different glycosyldonor/acceptor pairs react. The order in which different donor/acceptorpairs react will depend, in turn, on the relative reactivity ofdifferent donor/acceptor pairs. The relative reactivity ofdifferent/donor acceptor pairs can be manipulated in various ways, asalready described above (e.g., by manipulating the structure of thesulfoxide groups used and by protecting some glycosyl acceptors withsilyl ethers to slow down the rate at which they react).

Other factors that influence the relative reactivity of glycosyl donorsand acceptors, such as the presence of electronegative protecting groupson the sugar rings or the presence of steric hindrance can also beexploited. See, e.g., Binkley Modern Carbohydrate Chemistry, MarcelDekker, Inc: New York, 1988; also, Paulsen Angew. Chem. Int. Ed. Engl.1982, 22, 156. Hence, potentially many factors can be taken into accountin the implementation of the disclosed method of forming multipleglycosidic linkages to produce chemically diverse mixtures.

5.4. Catalytic Activation of Anomeric Sulfoxides

Elsewhere in this disclosure, the triflic anhydride activation ofanomeric sulfoxides was described, and the mechanism of thisglycosylation reaction discussed. Triflic anhydride reacts with thesulfoxide to form a trifloxy sulfonium salt that is extremely reactive.

In the presence of base, half an equivalent of triflic anhydride wassufficient to activate a full equivalent of sulfoxide. The phenyltrifluromethanesulfenate (PhSOTf) generated during the course of thereaction evidently activated the remaining 0.5 equivalent of the sugarsulfoxide (See, below). Indeed, others have used sulfonate esters toactivate thioglycosides. For example, Ogawa et al. use phenyl seleniumtriflate to activate phenyl and alkyl thioglycosides. See, Ito and OgawaTetrahedron Lett. 1987, 28, 2723. ##STR4##

In the absence of base, however, less than 0.05 equivalents of triflicanhydride activated a full equivalent of sulfoxide. Because the trifloxyphenyl sulfonate (PhSOTf) and triflic anhydride combined did not amountto more than 0.1 equivalent, some other species generated in thereaction was activating the sulfoxide in a catalytic cycle. It wasreasoned that the catalyst in question was triflic acid (TfOH). TFOH hasbeen used by others to activate other glycosyl donors. See, e.g.,, mostrecently, Lonn Glycoconjugate J. 1987, 4, 117; Mootoo et al. J. Am.Chem. Soc. 1989, 111, 8540; Evans et al. J. Am. Chem. Soc. 1990, 112,7001; and Veeneman et al. Tetrahedron Lett. 1990, 31, 1331. Furthermore,it was discovered that only catalytic amounts are typically requiredbecause the acid is regenerated in the reaction.

To determine whether TfOH can activate anomeric sulfoxides, thefollowing experiment was conducted using perbenzylated glucose sulfoxideI as the glycosyl donor and the C-6 primary alcohol 2a as the glycosylacceptor (See, scheme below).

The sulfoxide I (1.5 equivalents) was treated with triflic acid (0.05equivalents) at -78° C. in methylene chloride. This step was followed bythe addition of the nucleophile (1.0 equivalent) to the reaction. All ofthe sulfoxide was consumed to form product, indicating that triflic acidin catalytic amounts activates anomeric sulfoxides.

5.4.1. Mechanism

Although not wishing to be limited by theory, the following mechanisticinterpretation is offered for the benefit of the interested reader. Thestereochemical outcome for glycosylation using the catalytic triflicacid method was identical to that obtained from the stoichiometrictriflic anhydride method. It was surmised that both reactions proceedvia the same reactive intermediate, i.e., an oxonium ion or tight ionpair.

With the TFOH method, however, the yield for the desired disaccharide 3was low. A significant amount of lactol, 4, and the 1,1-dimer, 5, wereproduced as by products (See, below). The nature of these byproductsindicated to the present applicant that water was present in thereaction. In particular, if an oxonium ion is trapped by water, a lactolwill be formed. If the anomeric lactol then traps another oxonium ion, a1,1-dimer of the glycosyl donor would be formed. ##STR5## R═H with Tf₂O; R═Si(CH₃)₃ with TfOH

To prevent the formation of water, the glycosylations were conductedunder scrupulously anhydrous conditions, using activated molecularsieves. Despite these precautions however, formation of byproducts,accounting for 40% of the mass balance, was observed. This lastobservation suggested that water was somehow being formed during thecourse of the reaction, possibly from the disproportionation of phenylsulfenic acid. ##STR6##

As illustrated in the scheme, above, the first step of the catalyticcycle is protonation of the sulfoxide to form a sulfonium salt. Thesulfonium salt then ejects phenyl sulfenic acid (PhSOH) to form anoxonium ion or a tight ion pair. The nucleophile traps the oxonium ion,subsequently regenerating TfOH. In every catalytic cycle, one moleculeof sulfoxide forms product and generates one molecule of phenyl sulfenicacid as byproduct.

Sulfenic acids are a class of organo sulfur compounds that have eludedisolation because of their instability. They have highreactivity as bothelectrophiles and nucleophiles. Sulfenic acids readily undergodisproportionation to thiosulfinate esters and water. The postulatedmechanism for disproportionation, incorporating their dualelectrophilic/nucleophilic character, is illustrated below. ##STR7##

5.4.2. Addition of Scavengers for Sulfenic Acids

Sulfenic acids readily add to electron deficient alkenes and alkynes toform vinyl sulfoxides. Thus, it may be possible to trap these compoundswith a sulfenic acid scavenger before they self condense. Examples ofalkenes and alkynes frequently used to trap sulfenic acids includemethyl propionate, methyl propiolate, styrene, and dimethyldicarboxylate. The above compounds were screened as potentialscavengers, and methyl propiolate was found to be the most effective.

In a typical reaction, 1 and 2 were allowed to react with TfOH in thepresence of methyl propiolate (20 equivalents). The yield for thereaction improved from 35% (in the absence of methyl propiolate) to 45%(in the presence of methyl propiolate). Although the reaction yieldimproved, significant quantities of 4 and 5 were still produced. Hence,still further ways to prevent the disproportionation of sulfenic acidwere sought.

5.4.3. Use of Silyl Ethers as Nucleophiles

It was reasoned that the use of silyl ether protected alcohols asnuclephiles could further minimize the buildup of water. The silylethers could react under mild reaction conditions to produce the desireddisaccharide condensation product, TMSOTf and phenyl sulfenic acid. Thesulfenic acid could then be expected to react with TMSOTf to form thesilyl phenyl sulfonate (PhSOSi(CH₃)₃), thereby regenerating triflic acid(See, below). It was reasoned that because silylated sulfenates are muchmore stable than sulfenic acids and can be expected not todisproportionate as readily, silylated sulfenates could help minimizewater build-up. See, Nakamura J. Am. Chem. Soc. 1983, 105, 7172.##STR8##

Accordingly, perbenzylated glucose sulfoxide 1 (1.5 equivalents) wastreated with triflic acid (0.05 equivalents) in methylene chloride at-78° C. The silyl ether of the nucleophile (2b) was added to thereaction. After work-up the desired trisaccharide 3 was isolated as themajor product in 60% yield. (See, first scheme in Section 5.4.1.) Thus,by using the silyl ether of the nucleophile, the reaction yield improveddramatically, i.e., from 35% to 60%.

5.4.4. Application of the Catalytic Method for the Synthesis of 2-DeoxyOligosaccharides

In subsequent investigations the scope of the catalytic triflic acidmethod for activating sulfoxides was explored. Table I shows acomparison of the catalytic triflic acid and stoichiometric triflicanhydride methods for glycosylation using a range of 2-deoxy glycosylsulfoxides as glycosyl donors.

2-Deoxy glycosyl sulfoxides are notoriously unstable and tend to givelow yields of coupled product (See, Table I). The stoichiometric triflicanhydride method for activating sulfoxides does not always give goodresults with 2-deoxy glycosyl donors. The catalytic triflic acid methodhowever, works very well, presumably because the mild reactionconditions minimizes decomposition of 2-deoxysulfoxides. In fact, theuse of triflic acid improves the glycosylation yields by at least 50%for all the cases examined.

                                      TABLE I                                     __________________________________________________________________________    Synthesis of 2-Deoxy Glycosides                                                                                   Yield Ratio                               Entry                                                                            Glycosyl acceptor.sup.a                                                                     Glycosyl donor                                                                              Glycoside                                                                          Ti.sub.2 O                                                                       TfOH                                                                             (α:β)                    __________________________________________________________________________        ##STR9##                                                                                    ##STR10##    7    50%                                                                              88%                                                                              5:1                                     ##STR11##    6             9    40%                                                                              75%                                                                              >20:1                               3. 2                                                                                            ##STR12##    11   40%                                                                              85%                                                                              1:2                                     ##STR13##                                                                                   ##STR14##    14   57%                                                                              80%                                                                              1:2                                     ##STR15##                                                                                   ##STR16##    17   35%                                                                              60%                                                                              >20:1                               __________________________________________________________________________     .sup.a R = H, with triflic anhydride; R = OSi(CH.sub.3).sub.3, with           triflic acid.                                                            

As can be seen from the results listed in Table I, the yields obtainedfrom the catalytic glycosylation method are comparable to the bestyields reported in the literature for the various glycal methods.Furthermore, the catalytic TfOH can be used to activate sulfoxides evenin the presence of acid sensitive functional groups (Table I, Entry 2).

5.5. Aspects of the Catalytic TfOH and the Stoichiometric Tf₂ O Methodsfor Activating Sulfoxides

The two glycosylation methods complement each other. The catalytictriflic acid method is advantageous when the sulfoxide is unstable. Thereaction conditions are mild; therefore, decomposition of glycosyldonors is minimized. Furthermore, side reactions such as triflation orsulfenylation of the nucleophile, which can result in decreased yieldsfor glycosylation, do not occur with the catalytic TfOH method. However,the catalytic triflic acid method for activating sulfoxides is alsosignificantly slower and requires slightly higher temperatures (-78° C.to -30° C.) compared to the triflic anhydride method. Additionally, thecatalytic method is not efficient when electron-withdrawing protectinggroups are present on the glycosyl donor.

The stoichiometric Tf₂ O glycosylation method, on the other hand, worksextremely well for glycosyl donors with electron-withdrawing protectinggroups. It may be the best glycosylation method available whenneighboring group participation is used to obtain stereoselectivity.

An important point to note is that neither triflic acid nor triflicanhydride activate anomeric phenyl sulfides under the reactionconditions used (Table I, Entry 5). Since anomeric sulfides can bereadily converted to the sulfoxides under extremely mild conditions(mCPBA, CH₂ Cl₂, -78° C. to 0° C.) both methods lend themselves readilyto iterative strategies for oligosaccharide synthesis.

It has, thus, been shown that anomeric sulfoxides can be activated forglycosylation with a catalytic quantity of a strong organic protic acid,such as triflic acid. The glycosylation reaction proceeds under verymild conditions and offers the following advantages: (i) decompositionof sulfoxides is minimal under the reaction conditions; and (ii)problems of triflation and sulfenylation of the nucleophile areeliminated. These advantages are significant, especially in the contextof solid phase oligosac-charide synthesis where sulfenylation ortriflation of the nucleophile can result in the capping of a growingoligosaccharide chain on a resin and, hence, termination of thesynthesis.

This catalytic method complements the triflic anhydride method, and isespecially useful for the construction of 2-deoxy oligosaccharides. Infact, the catalytic triflic acid method has been employed in anefficient construction of the 2-deoxy trisaccharide of ciclamycin 0, asdescribed elsewhere in the present disclosure. Finally, we havedemonstrated that neither triflic anhydride nor triflic acid activatesanomeric phenyl sulfides under the reaction conditions used; therefore,both methods readily lend themselves to iterative strategies foroligosaccharide synthesis.

5.6. Application of the Single-Step Glycosylation Method to theSynthesis of an Anthracyline Antibiotic

We have found that the order of reactivity of different anomeric phenylsulfoxides can be controlled by varying the substituents at the paraposition of the phenyl ring. Consequently, we have succeeded insynthesizing ciclamycin 0 trisaccharide to be synthesizedstereoselectively in 25% yield from the component monosaccharides in onestep. The synthetic approach is outlined in FIG. 15.

Salient features of the synthesis include employing a catalytic triflicacid glycosylation method to construct all the 2-deoxy glycosidic bondsstereoselectively. Also, the trisaccharide bears a phenyl sulfide at theanomeric center of the A ring. Anomeric phenyl sulfides are stable("disarmed") to the conditions that activate anomeric sulfoxides forglycosylation. They can be readily oxidized under mild conditions. Thusthe sulfoxide glycosylation method lends itself well to an iterativestrategy for oligosaccharide synthesis. The sulfide on the A ring of theciclamycin trisaccharide was oxidized to the corresponding sulfoxidewith mCPBA and then coupled to the aglycone.

It has been discovered that the rate limiting step of thesulfoxide-mediated glycosylation reaction is triflation of thesulfoxide. The reactivity of phenyl sulfoxides can therefore bemodulated by varying the substituents in the para position of the phenylring. The observed reactivity trend is as follows:

    p-OMe>p-H>p-NO.sub.2

Hence, when perbenzylated glucose para-methoxy phenyl sulfoxide 27 (2.0eq) and perbenzylated glucose phenyl sulfoxide 28 (2.0 eq) were premixedtogether in CH₂ Cl₂ and treated with triflic anhydride (1.0 eq), base(2.0 eq) and the nucleophile 29 (2.0 eq) at -78° C., it was observed byTLC that the para-methoxy phenyl sulfoxide was activated selectively.##STR17##

The products isolated after chromatography included the disaccharide(80%) and unreacted phenyl sulfoxide (<60% yield). However, when thesame reaction was conducted in the presence of excess triflic anhydride,both the sulfoxides 27 and 28 were activated, presumably in a sequentialmanner, to give the glycosylated product.

Moreover, additional competition experiments revealed that the relativereactivity of glycosyl acceptors (nucleophiles) can also be manipulated.Thus, perbenzoylated 2-deoxy fucose phenyl sulfoxide 32 (2.0 eq), thenucleophile 31a (1.0 eq), the silyl ether 31b (1.0 eq), and the base(2,6-di-tert-butyl-4-methyl pyridine, 2.0 eq) were premixed in CH₂ Cl₂and cooled to -78° C. This reaction mixture was then treated withtriflic anhydride (1.0 eq). The reaction was followed by TLC whichindicated that the sulfoxide 32 and the nucleophile 31a were consumed(to form the disaccharide in 60% yield) while the silyl ether 31bremained unreacted. ##STR18##

In another experiment, an excess of the sulfoxide 32 (5.0 equiv) wasused; in this case, the nucleophile 31a was consumed first followed bythe silyl ether 31b. Thus, silyl ethers react more slowly thanunprotected alcohols as glycosyl acceptors, presumably, because theymust first be unmasked.

Having demonstrated that the ability to manipulate the reactivity ofboth the glycosyl donors and the glycosyl acceptors, the synthesis ofthe ciclamycin trisaccharide was pursued according to FIG. 15. It washoped that the p-methoxy phenyl sulfoxide B would be activated first,allowing it to react with the C-4 alcohol of A to form the ABdisaccharide. Then the phenyl sulfoxide C would be activated and couplewith the AB disaccharide to form the desired trisaccharide ABC.

5.6.1. One-Step Synthesis of the Trisaccharide

The sulfoxides 21, and 22 and the nucleophile 23 were premixed anddissolved in a 1:1 mixture of ether-methylene chloride at -78° C. Methylpropiolate (20 eq), followed by a catalytic quantity of triflic acid,(0.05 eq) were added to this solution. The reaction was stirred at -70°C. for half an hour and then quenched by pouring it into a saturatedsodium bicarbonate solution. ##STR19##

The desired trisaccharide 49 was the major product isolated in 25% yieldafter flash chromatography. No other trisaccharides were isolated fromthe reaction mixture. The only other significant coupled productisolated was the disaccharide 48, the precursor to the trisaccharide.The reaction had taken place in a sequential manner as hoped with thep-methoxyphenyl sulfoxide B becoming activated first and then reactingwith the free C-4 hydroxyl of nucleophile A to form the AB disaccharide(48). Subsequently, the phenyl sulfoxide C is activated and reacts withthe disaccharide AB to form the desired trisaccharide ABC (49).

In keeping with the proposed mechanism, when the same reaction isconducted at -100° C. (hexane-liquid nitrogen bath), the productsisolated are the silyl ether 47 of the disaccharide AB (60% yield) alongwith the unactivated sulfoxide C. Performing the experiment at lowtemperature, thus, confirms the stepwise nature of the reaction.

The yield of the one step glycosylation is limited not by any undesiredcross coupling but by the instability of the glycosyl donors,particularly the keto sulfoxide C, which decomposes readily at roomtemperature even in the absence of activating agent. Indeed, less than5% of the disaccharide from the cross coupling of phenyl sulfoxide 21and free alcohol 23 was detected even though 21 is present in excess; nodisaccharide from the cross coupling of 21 and 22 was detected. Thepresence of the ketone functional group in the pyranose ring maycontribute to the instability of this sulfoxide.

In an effort to increase the overall yield for the glycosylation, theuse of a suitably protected form of the keto sulfoxide was explored.

5.6.2. Improving the BC Coupling Yields

Since the AB disaccharide 48 and the nucleophile 23 are structurallyvery similar, the nucleophile 23 was chosen as a model compound for theglycosyl acceptor in the glycosylation reaction with C. The precursor tothe keto sulfide, the C-4 equatorial alcohol, was chosen as the glycosyldonor. The effect of different protecting groups at the C-4 center wereexamined. ##STR20##

The use of a suitably protected C-4 alcohol of the glycosyl donorimproved the yield for the glycosylation dramatically (40-60%). For allof the cases examined, however, there was loss of stereochemical controlat the anomeric center, as illustrated in Table II, immediately below.

                  TABLE II                                                        ______________________________________                                        Effect of Protecting Groups at C-4                                            Protecting group                                                                              Yield of    Ratio                                             R (51)          disaccharide (52)                                                                         α:β                                    ______________________________________                                        CH.sub.3 CO     40%         1:2                                               pMeOC.sub.6 H.sub.5 CH.sub.2                                                                  60%         1:2                                               TBDMS           60%         1:1                                               TBDPS           60%         1:1                                               ______________________________________                                    

The presence of a suitably protected C-4 axial hydroxyl on the C ringwas also examined. In this case, the desired α stereoselectivity wasobtained; however, two additional steps following glycosylation wererequired. These include deprotection of the C-4 alcohol followed byoxidation of the axial alcohol to the ketone. These additional stepsresult in decreasing the overall yield. Thus, although a 25% yield forthe one step synthesis of the trisaccharide appears modest, lack offurther functional group manipulations makes the synthesis efficient.

5.6.3. Coupling of Trisaccharide to the Aglycone ε-Pyrromycinone

The trisaccharide has an anomeric phenyl sulfide on the A ring. Thissulfide was oxidized to the sulfoxide using mCPBA. The aglyconeε-pyrromycinone 15 (1.0 eq) and the trisaccharide sulfoxide 50 (3.0 eq)were dissolved in a 1:1 mixture of ether-methylene chloride and cooledto -78° C. Methyl propiolate (20 eq) was added to the reaction mixture,followed by a catalytic quantity (0.05 eq) of triflic acid.

A TLC taken soon after the addition of triflic acid indicated thepresence of a new spot just above the aglycone. After work up andpurification by chromatography, this new compound was identified by NMRspectroscopy to be the aglycone coupled to the trisaccharide (54). TheJ_(H-H) coupling constant of 3.0 Hz for the anomeric proton wasconsistent with α stereochemistry of the glycosidic linkage.

5.6.4. Deprotection of Ciclamycin

To complete the synthesis of ciclamycin 0 the removal of the benzylether protecting groups on the A and B ring was required. The benzylethers were removed by hydrogenolysis using Pd(OH)₂ on carbon as thecatalyst. Unfortunately, under these reaction conditions, in addition tothe benzyl ethers, the aglycone also gets cleaved. In retrospect, thiswas not surprising since the C-7 hydroxyl of the aglycone to which thesugar is attached resembles a benzyl ether. Thus, to obtain the intactciclamycin 0, hydrogenolysis conditions could not be used. To circumventthis problem, the protecting groups on the sugar rings A and B needed tobe changed.

Para methoxy benzyl ethers can be readily cleaved under mild oxidativeconditions using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). See,for example, Ikemoto and Schreiber J. Am. Chem. Soc. 1990, 112, 9657;Horita et al. Tetrahedron, 1986, 42, 3021; Oikawa et al. Tet. Lett.1984, 25, 5393; and Carbohydrates, Ed. Collins, P. M. Chapman and Hall:New York, 1987. Thus, a 1:1 mixture of marcellomycin (which bears atrisaccharide at the C-7 position of the aglycone) and the A ring (withp-methoxy benzyl ether protecting group at C-3) were treated with anexcess of DDQ. Under these reaction conditions only the p-methoxy benzylether on the A ring was hydrolyzed while the marcellomycin remainedintact. Based on this result, the protecting groups on the ciclamycintrisaccharide may preferrably be changed from benzyl to p-methoxy benzylethers. ##STR21##

The monosaccharides 21, 22a and 23a can be used to synthesize thedesired ciclamycin trisaccharide in one-step (20% yield) following theusual procedure. The trisaccharide sulfide was oxidized to the sulfoxidewith mCPBA and then coupled to the aglycone using the catalytic triflicacid glycosylation method.

The deprotection of the coupled product 54a required the removal of thepara-methoxy benzyl ethers. The coupled product 54a (1 mg) was treatedwith DDQ in CH₂ Cl₂ and stirred at room temperature for 10 hours. Thereaction proceeded cleanly to give ciclamycin 0 quantitatively.

5.7. Rapid Synthesis of Oligosaccharides Through ControlledPolymerization: Constrained Libraries of 2-Deoxy Fucose Homopolymers

The 2-deoxy fucose sulfoxide B used for the one step synthesis of theciclamycin trisaccahride is a bifunctional sugar. It contains a leavinggroup at the anomeric center (p-methoxy phenyl sulfoxide) and can serveas a glycosyl donor. In addition, it has a silyl ether at the C-4 centerand can also serve as a glycosyl acceptor.

The most common 2-deoxy sugars found in bioactive natural products are2,6-dideoxy sugars. They frequently occur as dimers or trimers attachedto an aglycone. Given our success with the synthesis of a complicatedtrisaccharide in one step, we wondered if it was possible to extend thisidea for the rapid assembly of oligosaccharides through controlledpolymerization. The bifunctional 2,6-dideoxy B ring presented anopportunity to explore the possibility of synthesizing homopolymers in asingle reaction. ##STR22##

The sulfoxide 22 (2.0 eq) and the nucleophile 55 (1.0 eq) were premixedin 1:1 mixture of ether-methylene chloride and cooled to -78° C. Thereaction mixture was treated with base (2.0 eq) and triflic anhydride(1.0 eq) (See, above). A TLC of the reaction taken soon after indicatedthe presence of two new spots related to product. Afterchromatographytwo products were isolated and characterized by NMRspectroscopy. These products were the AB disaccharide 56 (45% yield) andthe ABB trisaccharide 57 (20% yield). The J_(H-H) coupling constants forthe anomeric protons were consistent with e stereochemistry for all theglycosidic linkages. This result indicated that homopolymers of 2-deoxyfucose can be formed stereoselectively in one step.

To determine whether higher order polymers of 2-deoxyfucose could beobtained, the number of equivalents of the sulfoxide B (22) used in theglycosylations was increased. ##STR23##

When 5.0 equivalents of B and 1.0 equivalent of A were used, astatistical mixture of di, tri, tetra, penta and hexasaccharides wasobtained in a single reaction, as noted above. Thus, using the sulfoxidemethod it is possible to rapidly synthesize a mixture of homopolymers of2-deoxy fucose through controlled polymerization.

The sulfoxide glycosylation method is powerful and versatile. It can beused to rapidly synthesize a complicated trisaccharide like ciclamycinfrom component monosaccharides in a single reaction. This strategy couldbe extended to synthesize a hexasaccharide from the componentdisaccharides in one step. In addition, by using bifunctional sugars itis possible to synthesize a mixture of homopolymers (of varying chainlength) in a single step with the sulfoxide method. Thus far, we haveonly examined 2-deoxy fucose as a substrate for the controlledpolymerization reactions. Nevertheless, this strategy could in principlebe extended to include other bifunctional substrates as well. ##STR24##

By using a combination of the one-step and controlled polymerizationstrategy it is possible to very rapidly synthesize libraries ofoligosaccharides with various aglycones attached, as shown above. Theseoligosaccharide libraries can be screened for DNA binding, for example,by using DNA affinity chromatography. Such a study will help elucidatethe features in oligosaccharides that confer DNA binding.

The sulfoxide glycosylation method is rapid, flexible and efficient forthe construction of oligosaccharides using conventional approaches. Thereactivity of glycosyl donors can be modulated by varying thesubstituent at the para position of the phenyl ring. Electron-donatingsubstituents accelerate the rate of the reaction relative to theunsubstituted case, while electron withdrawing groups decelerate therate of the reaction. The reactivity of glycosyl acceptors can bemodulated as well. Silyl ethers react more slowly in glycosylations thanfree alcohols. This permits the controlled formation of two or moreglycosidic linkages in a single reaction. This strategy was employed tosynthesize the ciclamycin 0 trisaccharide stereoselectively fromcomponent monosaccharides in a single step. Furthermore, bifunctionalsugars can be used to synthesize libraries of oligosaccharides. Thesulfoxide method is thus a versatile glycosylation method that allowsthe rapid synthesis of oligosaccharides through controlledoligomerization.

5.8. Formation Of Glycosidic Linkages On The Solid Phase

A potential glycosyl acceptor is attached to an insoluble support(hereafter termed the resin) through a linkage that can be readilycleaved at the end of the synthesis using conditions that do not damageglycosidic linkages. The resin may be any insoluble polymer that swellsin organic solvents and has sites for attaching the glycosyl acceptor.Preferred resins include, but are not limited to, polystyrene resins,such as the Merrifield resin, and PEG-derivatized polystyrene resins,such as the TentaGell resins.

The type of linkage depends on the type of functional sites available onthe polymer phase and on the glycosyl acceptor. Becausepolystyrene-based resins can be readily functionalized with chloromethylsubsitituents, the linkage is typically a benzyl ether, formed bynucleophilic displacement of a benzyl chloride on the resin with a freehydroxyl on the glycosyl acceptor. Alternatively, a benzyl ester can beused which is formed by nucleophilic displacement of a benzyl chlorideoil the resin with the salt of an acid on the glycosyl acceptor. (FIG.4) Both types of linkages can be readily hydrolyzed at the anomericcarbon of the glycosyl acceptor by treating the resin with Hg(II)compound. Alternatively, the ester linkage can be hydrolyzed bymethanolysis as is done for ester linkages to resins in peptidesynthesis. The Hg(II) method is preferred for treating aliquots of theresin to monitor the extent of reaction. The Hg(II) method is alsopreferred when the lactol of the completed oligosaccharide is desired asa final product. The methanolysis method is preferred when the sulfideof the completed oligosaccharide is desired as a final product (FIG. 4).

The potential glycosyl acceptor may be any molecule having one or morepotentially reactive nucleophiles, including potentially reactivehydroxyls, amines, and/or thiols, provided that it also has a suitablesite for attachment to the resin. A potentially reactive nucleophile isa free nucleophile or a nucleophile with a temporal protecting groupthat can be removed readily once the glycosyl acceptor is attached tothe resin. The potential glycosyl acceptor may also have permanentlyprotected nucleophiles, which are nucleophiles that cannot bedeprotected under the conditions that are used to remove the temporalprotecting groups. The potential glycosyl acceptor may be a sugar orsome other nucleophile-bearing molecule, including, but not limited to,steroids, amino acids or peptides, polar lipids, polycyclic aromaticcompounds, and the like. Protecting group schemes for sugars that permitselective protection and deprotection at any position are wellknown.See, e.g., Binkley, above.

Following attachment to the resin, the potentially reactive nucleophileis selectively deprotected, if necessary, and the derivatized resin islyophilized overnight and stored in a desiccator until use. The resin isthen preferably placed in a specially designed reactor vessel with aglass frit. Any openings are sealed, e.g., with rubber septa (See, e.g.,FIG. 5). There may be many variations on the general apparatus. However,the important features can be enumarted as follows:

a) An inlet for the addition of solvent and dissolved reagents to thereaction chamber and which is suitable for maintaining an anhydrousatmosphere; (In the apparatus shown, a rubber septum over a cup-shapedopening permits the addition of solvent and dissolved reagents by canulaor syringe needle while preventing exposure of the reaction chamber tothe outside air. In a preferred embodiment of the reaction vessel, thisinlet is also equipped with a T-connector or similar adapter whichallows the inlet to double as a vent for releasing inert gas, such asnitrogen or argon, to prevent the build up of excess pressure within theapparatus.)

b) A reaction chamber for holding the resin and reagent solution whichis equipped with a frit or filter of such coarseness or porosity so thatunbound components, such as unreacted dissolved reagents, but not resin,can be washed from the reaction chamber;

c) A port, located on the side of the frit which is opposite to theinlet side, for introduction of an inert gas; the gas passes through thefrit, thus agitating the reaction mixture, and settles over the reactionmixture, thus maintaining an anhydrous atmosphere inside the reactionchamber. (As evident from FIG. 5, the argon or nitrogen passes throughthe resin from below, opposing the flow of solvent through the frit andagitating the resin simultaneously. In a preferred embodiment, this portis equipped with a T-connector or similar adapter to allow the port tobe attached to an aspirator for removal of solvent under vacuum.)

It should also be noted that the configuration of the apparatus is suchthat the apparatus up to the level of most of the reaction chamber canbe immersed in a cooling bath. Hence, below the frit, the apparatus maybe in a U-shape, as shown in FIG. 5, so that the gas port can bepositioned above the cooling medium.

Next, an inert gas, such as argon or nitrogen, preferably argon, ispassed through the resin for about 1 hour. The resin is then suspendedin 3-5 mL anhydrous solvent including but not limited to toluene, ether,THF, methylene chloride, chloroform, propionitrile, or mixturesthereof). From the discussion in the previous section, it is understoodthat the choice of solvent will influence the stereochemical outcome ofglycosylation for reactions in which neighboring group participation isnot involved. The argon flow is adjusted to agitate the resin gently andprevent solvent from draining through the frit.

A glycosyl sulfoxide is then dissolved under anhydrous conditions in 2-4mL anhydrous solvent and transferred by canula to the reactor vesselcontaining the resin. The glycosyl sulfoxide may also have protectinggroups present elsewhere in the molecule. If the saccharide chain is tobe further extended, the glycosyl sulfoxide must also have at least onetemporal protecting group. Typically the glycosyl sulfoxide is added in2-4-fold excess relative to the amount of glycosyl acceptor on theresin.

Depending on the activation method used to initiate the glycosylationreaction, a non-nucleophilic base, such as 2,6-di-t-butyl-4-methylpyridine or Hunig's base (diisopropyl ethylamine), may be dissolved withthe glycosyl sulfoxide or added to the vessel containing the resin. Whenused, the base is preferably present in a slight excess relative to theamount of glycosyl sulfoxide added.

The reactor vessel containing the resin is then immersed in a cold bathat -78° C. To activate the glycosyl donors for reaction, either 0.05equiv. (i.e., catalytic) triflic acid or 0.5 equiv. of triflic anhydridediluted in a large volume of anhydrous solvent is added to the reactionmixture under anhydrous conditions. The molar equivalents are measuredrelative to the amount of glycosyl sulfoxide used. Also, dilution in alarge volume means that the volume of the neat activating agent isdiluted at least 100-fold by the addition of the appropriate volume ofsolvent (e.g., 1 μL of neat activating agent is added to at least 99 μLof solvent before addition to the donor).

The addition of activating agent may be carried out, for example, withthe aid of a canula. Other activating agents suitable in the presentmethod include, but are not limited to, an alkyl- or arylsilyl triflate(e.g., trimethylsilyl triflate), an alkyl- or arylsulfenyl triflate, andan alkyl- or arylselenenyl triflate. If protons are generated in thereaction (as when 0.5 equiv. of triflic anhydride is used to activatethe sulfoxide), an acid scavenger must be present in the resin mixture.Moreover, unless the activating agent is used in catalytic amounts(e.g., <0.1 equiv relative to glycosyl sulfoxide), the activating agentmust be diluted about 100-fold or more prior to addition. It has beendiscovered with triflic anhydride that dilution is critical, triflationof the glycosyl acceptors on the resin thus being avoided.

Next, the resin is gently agitated by the flow of argon. Typically thereaction is allowed to continue for approximately 30 minutes after whichthe resin is rinsed repeatedly to remove byproducts and unreactedglycosyl donor. If desired, the reaction may be monitored by removingaliquots of resin, rinsing the resin to remove reagents, and thenhydrolyzing the linkage to the resin. Alternatively, if the glycosylacceptor is a sugar which is attached to the resin via a sulfidederivative linked to the anomeric carbon, the link to the anomericcarbon may be hydrolyzed with a Hg(II) compound. Hydrolysis by Hg(II) ispreferred for monitoring the extent of the glycosylation reaction.

The products and the progress of the reaction may be analyzed by thinlayer chromatography using standards for comparison. For example, afterHg(II) hydrolysis of an aliquot from the reaction mixture, the solubleproducts are analyzed by TLC. The absence of the monosaccharide residuethat was bound to the resin is taken as an indication that the reactionhas proceeded to completion.

To obtain the products, the resin is typically rinsed repeatedly withmethylene chloride followed by methanol (preferably, about 10 cycles).The coupling may be repeated if necessary to drive the reaction tocompletion. Otherwise, if the saccharide chain is to be furtherextended, temporal protecting groups are next removed, the resin rinsedrepeatedly to remove reagents, and another glycosyl sulfoxide residueadded as before.

Upon completion of the synthesis and rinsing to remove reagents, thedisaccharide, oligosaccharide or glycoconjugate is removed from theresin. The product may then be purified and/or deprotected if desired.Alternatively, the disaccharide, oligosaccharide or glycoconjugate maybe used while still attached to the resin in screening procedures toelucidate biological activity.

Strategically, mixtures of oligosaccharides can also be produced bysolid phase synthesis and screened for biological activity. To producemixtures, more than one different type of glycosyl sulfoxide is added tothe resin at one or more cycles of the synthesis. It may be desirable tovary the sugars at only one position in the synthesis to probe thestructural requirements at that position. In this way,structure-activity relationships can be rapidly evaluated in cases whereboth a particular carbohydrate and its receptor are known.Alternatively, it may be desirable to vary the sugars at severalpositions in the synthesis, producing a complex mixture that can bescreened for binding to various receptors. In either case, if activityis detected, the active compound(s) can be identified using methodssimilar to those used in the peptide field for identifying activepeptides from mixtures produced by solid phase synthesis. See, forexample, Furka et al. Int. J. Peptide Protein Res. 1992, 37, 487; Lam etal. Nature 1991, 354, 82; Houghten, R. A. Nature 1991, 354, 84;Zuckermann et al. Proc. Natl. Acad. Sci. USA 1992, 89, 4505; PetithoryProc. Natl. Acad. Sci. USA, 1991, 88, 11510; Geyse et al. Proc. Natl.Acad. Sci. USA, 1984, 81, 3998; Houghten Proc. Natl. Acad. Sci. USA,1985, 82, 5131; Fodor et al. Science 1991, 251, 767.

Accordingly, it should be evident that the present invention is alsodirected to a U-shaped reaction vessel and a method of its use in asolid phase glycosylation reaction. The reaction vessel has proximal anddistal ends comprising (i) a reaction chamber equipped with a sealableinlet port at the proximal end, and (ii) a cylindrical compartmentforming a U-shape and equipped with a sealable outlet port at the distalend. The reaction chamber and cylindrical compartment are separated by aglass frit of a porosity such that the frit is capable of holding aninsoluble solid support while allowing soluble unbound components to bewashed away when desired from the insoluble solid support and allowingthe passage of gas for the agitation of the insoluble solid support.Preferably, the inlet and outlet ports are positioned above the reactionchamber to allow the immersion of the reaction chamber in a temperatureregulated medium while maintaining the inlet and outlet ports above themedium.

The reaction vessel thus is charged with the insoluble solid support,reagents, and reaction solvent required for performing a solid phaseglycosylation reaction. The inlet port is sealed and equipped with a gasvent, and the outlet port is sealed and equipped with a gas port. Theinert gas is introduced through the gas port at a flow effective toagitate the insoluble solid support and to oppose the flow of reactionsolvent through the frit. In specific embodiments, the gas ventscomprise a needle or cannula and one or both ports are equipped with aT-connector.

It should also be evident that the present invention is directed to acompound of the formula (IV): ##STR25##

in which each of the groups R₁, R₂, and R₃ can each independently be ahydrogen, lower alkyl or a hydroxyl protecting group, the group R₄ canbe a hydrogen, lower alkyl, a hydroxyl protecting group or the group OR₄together comprise at least a portion of a covalent linker moiety, andthe group N* is a masked group convertible to an amino group. Inspecific embodiments, the group R₁ is a hydrogen and the groups R₂ andR₃ singily or together comprise a hydroxyl protecting group (see, e.g.,the 4,6-O-benzylidene group of the compound 11, shown in FIG. 18). Thepreferred masked group N, comprises an azido group.

Also disclosed are certain intermediate compounds of the formula (V):##STR26##

in which the group R₁ can be a hydrogen, lower alkyl or a hydroxylprotecting group, the group R₄ can be a hydrogen, lower alkyl, ahydroxyl protecting group or the group OR₄ together comprises at least aportion of a covalent linker moiety, and the group N* is a masked groupconvertible to an amino group.

It is a further object of the present invention to provide a substanceof the formula (VI): ##STR27##

in which each of the groups R₁, R₂, and R₃ can independently be ahydrogen, lower alkyl or a hydroxyl protecting group, the group N* is amasked group convertible to an amino group, the group "Resin" comprisesan insoluble solid support, and the group X is covalent linker moiety.In specific embodiments of the present invention, the "Resin" comprisesa crosslinked styrene/divinylbenzene copolymer, such as a Merrifieldresin. Other suitable resins, include polystyrene resins orPEG-derivatized polystyrene resins.

The covalent linker moiety of X can be any group that allows thecovalent attachment of the glycosyl acceptor to the insoluble solidsupport. Such moieties may comprise, for example, aliphatic or aromaticether groups (--O--), carboxylic ester groups (--CO₂ --), amide groups(--CONH₂ --) or combinations of same (e.g., --OCH₂ CO₂ --). The maskedgroup, N*, can be any group that is stable to the conditions of thesolid phase glycosylation reaction but which can be converted to anamino group or derivative thereof, such as an acetylamino or aminesulfate group. Examples of suitable masked groups include a phthalimideor an azido group.

EXAMPLES

The following specific examples are provided to better assist the readerin the various aspects of practicing the present invention. As thesespecific examples are merely illustrative, nothing in the followingdescriptions should be construed as limiting the invention in any way.Such limitations are, of course, defined solely by the accompanyingclaims.

6.1. Synthesis Of The Ciclamycin 0 Trisaccharide In A Single Step

FIG. 1 illustrates one embodiment of the process for forming multipleglycosidic linkages in solution in which a specific trisaccharide, theciclamycin 0 trisaccharide, is synthesized stereospecifically inprotected form in a single step from the component monosaccharides. Themonosaccharides 1, 2, and 3 are combined in a ratio of 3:2:1, as shown(417 mg, 1.812 mmol; 541 mg, 1.2 mmol; and 165 mg, 0.604 mmol,respectively). Water is then removed from the mixture by distillation ofthe azeotrope from anhydrous toluene. (This drying step is carried outby dissolving the sugar mixture in toluene (ca. 30 mL) and removing thetoluene on a rotary evaporator under vacuum. The anhydrous sugar mixtureis then used directly or stored under inert gas until needed.)

The anhydrous sugar mixture is next dissolved in 20 mL of anhydrousmethylene chloride in a 50mL flame dried flask. Then, 20 mL of freshlydistilled diethyl ether containing 20 equivalents of methyl propiolateis added. (The propiolate ester is used to scavenge the sulfonic acidthat is produced in the reaction.) The solution is then cooled to -78°C. A catalytic amount of triflic acid (5.3 μL, 0.05 eq.) is then addeddropwise, and the reaction is allowed to warm from -78° to -70° C. overa period of 45 minutes and then quenched with saturated NaHCO₃. Thebiphasic mixture is then extracted with CH₂ Cl₂ (3×15 mL). The organicextracts are combined, dried over anhydrous Na₂ SO₄ and concentrated.The major product, trisaccharide 5, is isolated in 25% yield, based onmonosaccharide 3, after extraction with ethyl acetate and flashchromatography on silica gel (20% ethyl acetate/petroleum ether).

The ¹ H NMR spectra of trisaccharide 5 are shown in FIGS. 12 and 13. Thestereoselectivity achieved is a function of the donor-acceptor pairs andthe glycosylation conditions (solvent, temperature). We have found thatcatalytic triflic acid does not anomerize glycosidic linkages at anappreciable rate below -30° C. No other trisaccharide is produced.Indeed, the only other significant coupled product detected from thereaction is disaccharide 4 (Scheme 1 of FIG. 1, 15% yield; ¹ H NMR, FIG.14), the precursor to the trisaccharide 5. Less than 5% of thedisaccharide from the cross coupling of phenyl sulfoxide 1 and freealcohol 3 is detected even though 1 is present in excess; nodisaccharide from the cross coupling of 1 and 2 is detected.

Thus, the yield of trisaccharide 5 in the reaction is not limited by anyundesired cross coupling. However, the instability of the glycosyldonors, particularly keto sulfoxide 1, which decomposes readily at roomtemperature even in the absence of activating agent, can affect theyield.

The products of the reaction indicate that glycosylation takes place ina sequential manner, with p-methoxyphenyl sulfoxide Z activating fasterthan phenyl sulfoxide 1, and C-4 alcohol 3 reacting faster than C-4silyl ether 2. Consistent with this sequence, if the reaction isquenched at -100° C., only the silyl ether of disaccharide 4 can beisolated (60%).

Thus, the products of the one step reaction described above illustratethe principle established by the present invention; i.e. that thereactivity of both glycosyl donors and glycosyl acceptors can bemanipulated effectively so that glycosylation takes place in asequential manner to produce a desired oligosaccharide in a single step.

Finally, it should be noted that the trisaccharide (5) produced in theone step reaction has an anomeric phenyl sulfide on the A ring. Anomericphenyl sulfides are stable ("disarmed") to the conditions that activateanomeric phenyl sulfoxides for glycosylation, but they can be readilyoxidized under mild conditions. See, Mootoo et al. J. Am. Chem. Soc.1988, 110, 5583; Veeneman and van Boom Tet. Lett. 1990, 31, 275; andMehta and Pinto Tet. Lett. 1991, 32, 4435. Thus, the sulfoxideglycosylation reaction also lends itself well to an iterative strategyfor oligosaccharide synthesis. See, Friesen and Danishefsky J. Am. Chem.Soc. 1989, 111, 6656; Halcomb and Danishefsky J. Am. Chem. Soc. 1989,111, 6661; Mootoo et al. supra; Veeneman and van Boom, supra; and Mehtaand Pinto, supra; Nicolaou et al. J. Am. Chem. Soc. 1984, 106, 4189;Mootoo et al. J. Am. Chem. Soc. 1989, 111, 8540; Barrett et al. J. Am.Chem. Soc., 1989, 111, 1392. The ciclamycin trisaccharide 5 is oxidizedto the corresponding sulfoxide in 80% yield (1.2 eq. mCPBA, CH₂ Cl₂.-78°C. to -50° C., 2 hr) and is ready for coupling to the ciclamycinchromophore.

Monosaccharide 1 is prepared from L-rhamnose in 60% overall yield (¹ HNMR; FIG. 9). See, Martin et al. Carbohydr. Res. 1983, 115, 21.Monosaccharides 2 (¹ H NMR; FIG. 10) and 3 (¹ H NMR; FIG. 11) areprepared from L-fucose with overall yields of 47% and 52%, respectively.See, Giese et al. Angew Chem. Int. Ed. Engl. 1987, 26, 233.

According the another method of the present invention, the preparationof individual starting materials, their orchestrated condensation toform the trisaccharide of interest, and their subsequent coupling to anaglycone are described in greater detail, below.

6.1.1. Phenyl-3-O-benzoyl-2,6-dldeoxy-1-thio-α-L-galactopyranoside (31a)

The compound 37 (1.41 g, 5.9 mmol) is dissolved in dichloromethane andcooled to -78° C. under argon. Sodium hydride (566 mg, 23.6 mmol) isadded to this solution. There is brisk effervescence. After ten minutes,benzoyl chloride (2.05 mL, 17.7 mmol) is added to the solution. Thereaction is stirred at -78° C. for 1/2hour, gradually warmed -60° C. andthen quenched by pouring into saturated solution of NaHCO₃. Theresulting solution is extracted with CH₂ Cl₂ (3×50 mL); the organiclayers are combined, dried over anhydrous Na₂ SO₄ and concentrated undervacuum. Flash chromatography (20% ethyl acetate-petroleum ether) yieldsthe product as a white solid (1.6 g, 70%). R_(f) =0.3 (20% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 8.3 (m, 2H),7.8-7.2 (m, 8H), 5.85 (d, J=5.94 Hz, 1H, H-1), 5.55 (m, 1H, H-3), 4.7(q, J=6.60 Hz, H-5), 4.1 (bs, 1H, H-4), 2.78 (dt, J=5.93, 12.86 Hz, 1H,H-2_(ax)), 2.33 (dd, J=4.95, 13.19 Hz, H-2_(eq)), 2.12 (bs, 1H, OH),1.41 (d, J=6.60 HZ, 3H, CH₃).

6.1.2. Phenyl-3,4-O-benzoyl-2,6-dideoxy-1-thio-α-L-galaotopyranoside(32a)

This compound is synthesized fromphenyl-3,4-O-diacetyl-2,6-dideoxy-1-thio-α-L-galactopyranoside 36 in thefollowing two step sequence: (i) NaOMe/CH₃ OH, amberlite IR(120) plusion-exchange resin; and (ii) Benzoyl chloride, pyridine. R_(f) (TLC)=0.3(30% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ8.1-7.7(m, 6H), 7.5-7.2 (m, 9H), 5.71 (m, 1H, H-3), 5.61 (bs, 1H, H-4),5.48 (d, J=5.28 Hz, 1H), 4.33 (q, J=6.27 Hz, 1H, H-5), 2.45 (dt, J=3.63,12.54 Hz, 1H, H-2_(ax)), 2.17 (dd, J=4.95, 12.54 Hz, 1H, H-2_(eg)), 1.25(d, J=6.27 Hz, 3H, CH₃). 13C NMR (CDCl₃, 67.5 MHz) δ 169.49, 164.88,161.88, 134.11, 130.77, 130.00, 129.28, 129.13, 128.56, 128.44, 128.28,128.12, 127.83, 83.33, 69.93, 67.81, 65.74, 53.22, 30.62, 16.17.

6.1.3. Phenyl-3,4-O-benzoyl-2,6-dideoxy-1-sulfinyl-α-L-galactopyranoside(32)

The sulfide 32a (900 mg, 1.99 mmol) is dissolved in dichloromethane andcooled to -78° C. (acetone/dry-ice bath) under argon. mCPBA (483 mg,2.80 mmol) is added and the reaction is stirred at -78° C. for one hour.The reaction is gradually warmed to 0° C. over a period of two hours andthen quenched by pouring into saturated solution of NaHCO₃. Theresulting biphasic mixture is extracted with CH₂ Cl₂ (3×25 mL). Theorganic layers are combined, washed with brine and dried over anhydrousNa₂ SO₄. Flash chromatography (40% ethyl acetate-petroleum ether)provides the sulfoxide 32 as a white crystalline solid (690 mg, 80%yield). R_(f) (TLC)=0.4 (40% ethyl acetate-petroleum ether). ¹ H NMR(CDCl₃, 270 MHz) δ 8.1-7.65 (m, 6H), 7.5-7.2 (m, 9H), 5.9 (m, 1H, H-3),5.68 (d, J=2.64 Hz, 1H, H-4), 4.71 (d, J=5.61 Hz, 1H, H-1), 4.63 (q,J=6.27 Hz, 1H, H-5), 2.80 (dd, J=5.28, 14.19 Hz, 1H, H-2_(eq)), 2.55(dt, J=5.94, 12.53 Hz, 1H, H-2_(ax)), 1.27 (d, J=6.27 Hz, 3H, CH₃).

6.1.4.3,4-O-Benzoyl-2,6-dideoxy-galacto-pyranosyl-α-(1→4)-phenyl-3-O-benzoyl-2,6-dideoxy-1-thio-α-L-galactopyranoside (33)

The sulfoxide 32 (177 mg, 0.4 mmol), nucleophile 31a (80 mg, 0.2 mmol),silyl ether 31b (93 mg, 0.2 mmol) and base (82 mg, 0.4 mmol) arepremixed and azeotroped 3 times with toluene. To a 25mL flame-driedflask under argon is added freshly distilled CH₂ Cl₂. The azeotropedreactants are dissolved in 10 mL CH₂ Cl₂ and added to the flask, whichis then cooled to -78° C. After 10 minutes triflic anhydride (33.6 μL,0.2 mmol) is added. The reaction is followed by TLC (30% ethylacetate-petroleumether). The TLC indicates that the nucleophile 31a hasreacted completely while the silyl ether 31b remains unreacted. Thereaction is gradually warmed to -60 ° C. over a period of two hours andthen quenched by pouring into a saturated solution of NaHCO₃. Theresulting mixture is extracted with CH₂ Cl₂ (3×15 mL), dried overanhydrous Na₂ SO₄ and concentrated under vacuum. Flash chromatography(30% ethyl acetate-petroleum ether) gives the disaccharide 33 as a whitecrystalline solid (85 mg, 60% yield). R_(f) =0.4 (30% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 8.1-7.9 (m, 6H),7.6-7.1 (m, 14H), 5.80 (d, J=5.28 Hz, 1H, H-1), 5.74 (m, 1H, H-3), 5.47(m, 1H, H-3'), 5.43 (d, J=1.98 Hz, H-4), 5.21 (d, J=1.98 Hz, H-4'), 4.54(q, J=6.27 Hz, H-5), 4.39 (q, J=6.60 Hz, 1H, H-5'), 4.20 (d, J=2.31 Hz,1H, H-an), 2.96 (dt, J=5.61, 12.87 Hz, 1H, H-2'), 2.20 (m, 3H), 1.28 (d,J=6.60 Hz, 3H, CH₃), 0.47 (d, J=6.27 Hz, 3H, CH₃). ¹³ C NMR (CDCl₃, 67.5MHz) δ 165.95, 165.90, 165.69, 134.94, 133.42, 133.11, 132.92, 131.06,129.91, 129.88, 129.85, 129.76, 129.69, 129.57, 128.91, 128.58, 128.42,128.23, 127.01, 98.93, 83.79, 75.33, 70.34, 70.10, 67.60, 67.53, 65.71,30.98, 30.21, 17.41, 16.08.

6.2. Synthesis of the A Ring

The synthesis of the A ring starts with L-fucose, and is accomplished insix steps with an overall yield of 60%, according to the scheme, below.##STR28##

6.2.1. 1,3,4-O-Tri-O-acetyl-2,6-dideoxy-α-L-galactopyranoside (35)

To a solution of tetra-O-acetyl-fucose (5.0 g, 15.05 mmol) in 10 mL ofglacial acetic acid is added 15 mL hydrobromic acid (30%) in a dropwisemanner and the resulting solution stirred at room temperature. After twohours the reaction is complete. Work up is done under anhydrousconditions by pouring the reaction mixture into a flask containing 25 gof anhydrous sodium carbonate (Na₂ CO₃) suspended in 200mL of carbontetrachloride. The resulting mixture is stirred at room temperature for45 minutes and filtered. The procedure is repeated with the filtrate.The resulting solution is then concentrated under vacuum to afford crudebromide 34 (5.2 g, 80%). This is used without further purification inthe next step.

To a two-liter three-necked flask, equipped with a reflux condenser andan addition funnel, is added one liter of freshly distilled benzene. Thefucose bromide 34 (5.2 g, 12.17 mmol) is dissolved in benzene (15 mL)and added to the flask. The resulting mixture is heated to refux. AIBN(200 mg, 1.21 mmol) is added to this solution. After 30 minutes thetributyl tin hydride (4.91 mL, 18.25 mmol) in benzene (100 mL) is addeddropwise to the reaction mixture over a period of 16 hours via theaddition funnel. At the end of this time, the reaction mixture is thencooled to room temperature and concentrated under vacuum. Flashchromatography (25% ethyl acetate-petroleum ether) affords the product35 (2.5 g, 81%) as a crystalline white solid. R_(f) (TLC)=0.3 (25% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 6.24 (d, J=2.64 Hz,1H, H-1), 5.22 (m, 1H, H-3), 5.17 (t, J=0.66 Hz, 1H, H-4), 4.11 (q,J=5.94 Hz, 1H, H-5) 2.13 (s, 3H), 2.06 (s, 3H), 1.97 (s, 3H), 1.10 (d,J=6.6 Hz, 3H, CH₃); ¹³ C NMR (CDCl₃ 67.9 MHz) δ 170.13, 169.65, 168.82,91.51, 68.87, 66.93, 65.86, 28.37, 20.60, 20.42, 16.14, 16.01.

6.2.2. Phenyl-3,4-O-diacetyl-2,6-dideoxy-1-thio-α-L-galactopyranose (36)

Compound 35 (2.5 g, 9.11 mmol) and thiophenol (1.12 mL, 10.94 mmol) aredissolved in dichloromethane (100 mL) and cooled under argon to -78° C.Et₂ O.BF₃ (5.6 mL, 45.5 mol) is added dropwise via a syringe to thesolution. The reaction is kept at low temperature for one hour and thengradually warmed to 0° C. and quenched by pouring it into saturatedaqueous NaHCO₃. The resulting biphasic mixture is extracted with CH₂ Cl₂(3×50 mL); the organic layers are combined, dried over anhydrous Na₂ SO₄and concentrated. Flash chromatography of the crude material (15% ethylacetate-petroleum ether) gives the sulfide 36 as a white solid (2.1 g,71% yield). R_(f) (TLC)=0.3 (15% ethyl acetate-petroleum ether). ¹ H NMR(CDCl₃, 270 MHz) δ 7.5-7.2 (m, 5H), 5.73 (d, J=5.61 Hz, 1H, H-1), 5.29(m, 1H, H-3), 5.23 (bs, 1H, H-3), 4.56 (q, J=6.6 Hz, 1H, H-5), 2.49 (dr,J=5.94, 12.87 Hz, 1H, H-2_(az)), 2.38 (s, 3H, OAc), 2.06 (m, 1H,H-2_(eq)), 1.99 (s, 3H, OAc), 1.13 (d, J=6.6 Hz, 3H, CH₃). ¹³ C NMR forα anomer of sulfide (CDCl₃, 67.9 MHz) δ 170.51, 169.87, 159.54, 134.33,124.54, 114.54, 84.58, 69.69, 67.18, 65.51, 55.22, 20.79, 20.60, 16.34.

6.2.3. Phenyl-2,6-dideoxy-1-thio-α-L-galactopyranoside (37)

The compound 36 (2.1 g, 6.48 mmol) is dissolved in methanol (50 mL) andsodium methoxide (420 mg, 7.78 mmol) is added to the solution. Thereaction is stirred at room temperature for one hour. A TLC (40% ethylacetate-petroleum ether) taken at the end of this time indicates thatthe reaction is complete. The solution is neutralized with amberlite IR(120) plus ion-exchange resin (1.0 g). The solution is filtered througha fritted funnel, washed with ethyl acetate and concentrated to affordthe diol 37. This compound is used without further purification in thenext step.

6.2.4. Phenyl-3-O-benzyl-2,6-dideoxy-1-thio-α-L-galactopyranoside (23)

A solution of 37 (923 mg, 3.84 mmol) and dibutyl tin oxide (956 mg, 3.84mmol) in benzene (60 mL) is heated to reflux in a flask fitted with aDean-Stark apparatus. After 15 hours the reaction mixture is cooled toroom temperature and tetrabutylammonium bromide (1.24 g, 3.84 mmol) isadded followed by benzyl bromide (5 mL, 8.4 mmol). The resulting mixtureis refluxed further for two hours, then cooled to room temperature andconcentrated under vacuum. Flash chromatography on the crude product(15% ethyl acetate-petroleum ether) affords the sulfide 23 as a whitesolid (1.10 g, 90% yield). R_(f) (TLC) =0.5 (30% ethyl acetate-petroleumether). ¹ H NMR (CDCl₃, 270 MHz) α anomer δ 7.40-7.41 (m, 10H), 5.57 (d,J=5.61 Hz, 1H, H-1), 4.49 (s, 2H), 4.18 (q, J=6.6 Hz, 1H, H-5), 3.74 (m,1H, H-3), 3.70 (d, J=3.63 Hz, 1H, H-4), 2.20(dt, J=5.61, 12.5Hz, 1H,H-2), 2.14 (bs, 1H, OH), 1.95 (m, 1H, H-2'), 1.17 (d, J=6.6 Hz, 3H,CH₃). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 137.72, 135.20, 130.76, 128.84,128.52, 127.92, 127.67, 126.83, 83.93, 73.514, 70.13, 68.46, 66.83,30.67, 16.61. HRMS m/e 330.1290 (M⁺) calcd for C₁₉ H₂₂ O₃ S 330.1290.

6.3. Synthesis of the B ring

The synthesis of the B ring is accomplished as follows: ##STR29##

6.3.1.4-Methoxyphenyl-3,4-O-diacetyl-2,6-dideoxy-1-thio-L-galactopyranoside(38)

To a solution of 35 (1.95 g, 7.14 mmol) in distilled dichloromethane(100 mL) is added 4-methoxy thiophenol (1 mL, 8.56 mmol) and theresulting mixture is cooled to -78° C. To this is added Et₂ O.BF₃ (4.4mL, 35.70 mmol) dropwise. The reaction mixture is stirred a lowtemperature for 1/2hour then gradually warmed to -30° C. and quenched bypouring into a saturated solution of NaHCO₃. The resulting mixture isextracted with CH₂ Cl₂ (3×30 mL). The organic extracts are combined,dried over anhydrous Na₂ SO₄, filtered and concentrated under vacuum.The crude product is purified by flash chromatography (20% ethylacetate-petroleum ether) to afford the sulfide 38 (2.12 g, 85%). R_(f)(TLC)=0.4 (20% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz)δ 7.40 (d, 2H), 6.8 (d, 2H), 5.51 (d, J=5.61 Hz, 1H, H-1), 5.26 (m, 1H,H-3), 5.19 (t, J=2.97 Hz, 1H, H-4), 4.54 (q, J=6.6 Hz, 1H, H-5), 3.77(s, OCH₃), 2.32(dt, J=5.61, 12.80 Hz, 1H, H-2), 2.12 (s, 3H), 2.02 (dt,J=4.62, 12.80 Hz, 1H, H-2'), 1.98 (s, 3H), 1.07 (d, J=6.6 Hz, 3H, CH₃);¹³ C NMR (CDCl₃, 67.9 MHz) δ 170.51, 169.87, 159.54, 134.33, 124.54,114.54, 84.58, 69.69, 67.18, 65.51, 55.22, 30.24, 20.79, 20.60, 16.34;HRMS m/e 354. 1140 (M⁺), calcd for C₁₇ H₂₂ O₆ S 354.1137.

6.3.2. 4-Methoxyphenyl-2,6-dideoxy-1-thio-L-galacto-pyranoside (39)

To a solution of 38 (1.0 g, 2.82 mmol) in methanol (100 mL) is addedsodium methoxide (183 mg, 3.38 mmol). The reaction mixture is stirred atroom temperature for two hours and then neutralized by adding Amberliteresin (1.0 g). The reaction mixture is filtered and concentrated undervacuum to afford the diol 39 (760 mg, 100%). This is used withoutfurther purification in the next step. R_(f) (TLC)=0.1 (50% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) α anomer δ 7.35 (d,2H), 6.80 (d, 2H), 5.41 (d, J=5.28 Hz, 1H, H-1), 4.41 (g, J=6.6 HZ, 1H,H-5), 3.95 (m, 1H, H-3), 3.76(s, OCH₃) 3.65 (d, J=2.64 Hz, 1H, H-4),2.01(m, 2H, H-2, 2'), 1.23 (d, J=6.6 Hz, 3H, CH₃).

6.3.3. 4-Methoxyphenyl-3-O-benzyl-2,6-dideoxy-1-thio-L-galactopyranoside(40)

A solution of 39 (2.13 g, 7.90 mmol) and dibutyl tin oxide (1.96 g, 7.90mmol) in benzene (200 mL) is heated to reflux in a flask fitted with aDean-Stark apparatus. After 15 hours the reaction mixture is cooled toroom temperature and to it is added tetrabutyl ammonium bromide (2.54 g,7.90 mmol) followed by benzyl bromide (2.82 mL, 23.7 mmol). Theresulting mixture is refluxed further for two hours, then cooled to roomtemperature and concentrated under vacuum. Flash chromatography on thecrude product (15% ethyl acetate-petroleum ether) affords the sulfide 40as an oil (2.5 g, 88%). R_(f) (TLC) =0.25 (20% ethyl acetate-petroleumether). ¹ H NMR (CDCl₃, 270 MHz) δ 7.5-7.25 (m, 7H), 6.81 (d, J=8.91 Hz,2H), 5.49 (d, J=5.61 Hz, 1H, H-1), 4.6 (s, 2H), 4.32 (q, J=6.59 Hz, 1H,H-5), 3.85 (m, 1H, H-3), 3.82 (d, J=3.3 Hz, 1H, H-4), 3.77 (s, OCH₃),2.25 (dt, J=5.94, 12.8 Hz, 1H, H-2), 2.2 (bs, OH), 1.72 (m, 1H, ),H-2'), 1.27 (d, J=6.59 Hz, 3H, CH₃). ¹³ C NMR (CDCl₃, 67.9 MHz) δ159.25, 137.66, 134.03, 128.33, 127.70, 127.52, 124.98, 114.37, 84.81,73.40, 69.87, 68.35, 66.55, 55.06, 30.24, 16.47. HRMS m/e 360.1385 (M⁺),calcd for C₂₃ H₂₄ O₄ S 360.1396.

6.3.4.4-Methoxyphenyl-3-O-benzyl-2,6-dideoxy-1-thio-4-O-(trimethyl-silyl)-L-galactopyranoside(41)

A solution of 40 (1.4 g, 3.88 mmol) and triethylamine (1.62 mL, 11.64mmol) in dichloromethane (100 mL) is cooled to -78° C. under argon. Tothis solution is added TMSOTf (825 μl, 4.27 mmol) dropwise. The reactionis stirred at low temperature for 30 minutes and then quenched bypouring it into a solution of saturated NaHCO₃. The resulting mixture isextracted with CH₂ Cl₂ (3×30 mL). The organic extracts are combined,dried over anhydrous Na₂ SO₄ and concentrated. Flash chromatography (10%ethyl acetate-petroleum ether) afforded the product 41 (1.3 g, 83%) asan oil. R_(f) (TLC)=0.85 (15% ethyl acetate-petroleum ether). ¹ H NMR(CDCl₃, 270 MHz) δ 7.32 (d, J=8.58 Hz, 2H), 7.28 (m, 5H), 6.79 (d,J=8.91 Hz, 2H), 5.52 (d, J=5.28 Hz, 1H, H-1), 4.55 (d, J=0.99 Hz, 2H),4.21 (q, J=6.60 Hz, 1H, H-5), 3.79 (bs, 1H, H-4), 3.76 (s, OCH₃), 3.66(m, 1H, H-3), 2.32 (dt, J=5.61, 12.54 Hz, 1H, H-2), 1.97 (m, 1H, H-2'),1.14 (d, J=6.59 Hz, 3H, CH₃), 0.11 (s, 9H, TMS). ¹³ C NMR (CDCl₃, 67.9MHz) δ 138.19, 133.67, 128.15, 127.46, 127.39, 125.55, 114.32, 85.16,74.26, 71.05, 70.16, 67.76, 55.03, 30.11, 16.99, 0.5. HRMS m/e 432.1808(M⁺), calcd for C₂₃ H₃₂ O₄ SSi 432.1790.

6.3.5.4-Methoxyphenyl-3-O-benzyl-2,6-dideoxy-1-sulfinyl-4-O-(trimethyl-silyl)-L-galactopyranoside(22)

To a solution of the sulfide 41 (402 mg, 0.93 mmol) in dichloromethane(60 mL) is added an excess of solid sodium bicarbonate (1.0 g) and theresulting mixture cooled to -78° C. To this suspension is added mCPBA(193 mg, 1.11 mmol) and the resulting mixture is stirred at lowtemperature for 30 minutes. The temperature of the reaction mixture isgradually raised to 0° C. over a period of one hour and then quenched bypouring into a saturated solution of NaHCO₃. The resulting mixture isextracted with CH₂ Cl₂ (3×30 mL) the organic layers combined, dried overanhydrous Na₂ SO₄ and concentrated under vacuum. Flash chromatography(30% ethyl acetate-petroleum ether) affords the sulfoxide 22 (400 mg,96%) as a white solid. R_(f) (TLC)=0.4 (30% ethyl acetate-petroleumether). ¹ H NMR (270 MHz, CDCl₃) δ 7.51 (d, J=8.58 Hz, 2H), 7.4-7.2 (m,5H), 6.97 (d, J=8.58 Hz, 2H), 4.62 (d, J_(AB) =11.87 Hz, 1H), 4.55 (d,J_(AB) =11.88 Hz, 1H), 4.47 (d, J=5.27 Hz, 1H, H-1), 4.02 (q, J=6.6 Hz,1H, H-5), 3.91 (m, 1H, H-3), 3.84 (bs, 1H, H-4), 3.83 (s, OCH₃), 2.63(dd, J=4.62, 13.86 Hz, 1H, H-2), 2.02 (dt, J=5.61, 13.85 Hz, 1H, H-2'),1.12 (d, J=6.26 Hz, 3H, CH₃), 0.09 (s, 9H, TMS).

6.4. Synthesis of the C ring

The C ring is prepared as follows: ##STR30##

6.4.1. Methyl-4-O-acetyl-2,3,6-trideoxy-L-erythro-hexopyranoside (44)

To a solution ofMethyl-4-O-acetyl-2,3,6-trideoxy-L-erythro-hex-2-enopyranoside 43 (2.0g, 0.01 mmol) (See, Martin et al. Carbohydr. Res. 1983, 115, 21) inbenzene is added the catalyst Pd (OH)₂ /C (200 mg) and the resultingsuspension is shaken in a Parr shaker under H₂ (50 psi). After two hoursthe reaction mixture is filtered through Celite and concentrated undervacuum to give 44 (2.0 g, 100% yield). This is used in the next stepwithout further purification. R_(f) (TLC) =0.5 (20% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 4.63 (s, 1H, H-4),4.52 (bt, 1H, H-1), 4.32 (q, J=6.27 Hz, 1H, H-5), 3.22 (s, OCH₃), 2.02(s, 3H, OAc), 1.9 (In, 1H, H-3'), 1.85-1.65 (m, 3H, H-2, 2', 3), 1.02(d, J=6.27 Hz, 3H, CH₃). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 169.14, 96.78,72.96, 65.85, 53.70, 28.68, 23.69, 20.29, 17.34. HRMS m/e 187.0971 (M⁺)calcd for C₉ H₁₅ O₄ 187.0970.

6.4.2.Phenyl-4-O-acetyl-1-thio-2,3,5-trideoxy-β-L-arabino-hexapyranoside (45a)

To a solution of 44 (2.3 g, 12.23 mmol) and thiophenol (1.5 mL, 14.67mmol) in dichloroinethane (100 mL) cooled to -78° C. is added BF₃.OEt₂(4.5 mL, 36.69 mmol) dropwise. The reaction is stirred at lowtemperature for 30 minutes, gradually warmed to -60° C., and quenched bypouring into a saturated solution of NaHCO₃. The resulting mixture isthen extracted with CH₂ Cl₂ (3×25 mL); the organic layers are combined,dried over anhydrous Na₂ SO₄ and concentrated under vacuum. Flashchromatography (15% ethyl acetate-petroleum ether) affords the sulfide45a (3.0 g, 92%) as a white solid. R_(f) (TLC) =0.4 (15% ethylacetate-petroleum ether). ¹ H NMR (270 MHz, CDCl₃) δ 7.55-7.2 (m, 5H),5.52 (d, J=4.95 Hz, 1H, H-4), 4.59 (dt, J=4.62, 10.23 Hz, 1H, H-1), 4.30(m, 1H, H-5), 2.2-2.1 (m, 2H, H-3, 3'), 2.09 (s, 3H, OAc), 1.9-1.75 (m,2H, H-2, 2'), 1.17 (d, J=5.95 Hz, 3H, CH₃). HRMS m/e 266.0970 (M⁺) calcdfor C₁₄ H₁₈ O₃ S 266.0977.

6.4.3.Phenyl-4-O-hydroxy-1-thio-2,3,5-trideoxy-β-L-arabino-hexapyranoside(45b)

To a solution of 45a (3.0 g, 11.27 mmol) in methanol (100 mL) is addedsodium methoxide (365 mg, 6.76 mmol). The reaction mixture is stirred atroom temperature for two hours and then neutralized by adding Amberliteresin (2.0 g) and stirred for 15 minutes. The reaction mixture isfiltered and concentrated under vacuum to afford the alcohol 45b (2.52g, 100%). This is used without further purification in the next step.R_(f) (TLC)=0.2 (25% ethyl acetate-petroleum ether). ¹ H NMR (270 MHz,CDCl₃) δ 7.5-7.2 (m, 5H), 5.49 (d, J=4.62 Hz, 1H, H-1), 4.1 (m, 1H,H-4), 3.3 (m, 1H, H-5), 2.2-1.6 (m, 4H, H-2, 2',3, 3'), 1.2 (d, J=5.49Hz, 1H, CH₃).

6.4.4. Oxidation to Keto Sulfide (46)

A solution of oxalyl chloride (5.5 mL, 11.16 mmol) in dichloromethane(150 mL) is treated with DMSO (1.5 mL, 22 mmol) at -78° C. After 10minutes a solution of the alcohol 45b (2.27 g, 10.15 mmol) indichloromethane (15 mL) and triethylamine (7 mL, 50.75 mmol) is added tothe reaction mixture. The reaction mixture is stirred at low temperaturefor 30 minutes, then warmed to 0° C. and quenched by pouring it into asolution of NaHCO₃. The resulting mixture is extracted with CH₂ Cl₂(3×30 mL); the organic layers combined, dried over anhydrous Na₂ SO₄ andconcentrated under vacuum. Flash chromatography (15% ethylacetate-petroleum ether) afforded the ketone 46 (2.20 g, 97%) as a paleyellow oil. R_(f) (TLC)=0.35 (15% ethyl acetate-petroleum ether). ¹ HNMR (CDCl₃, 500 MHz) δ 7.65-7.20 (m, 5H), 5.52 (t, J=6.59, 6.96 Hz, 1H,H-1), 4.49 (q, J=6.6 Hz, 1H, H-5), 2.65-2.40 (m, 3H, H-3, 3', 2), 1.93(m, 1H, H-2'), 1.22 (d, J=6.59 Hz, 3H, CH₃). ¹³ C NMR (CDCl₃, 67.9 MHz)δ 209.58, 134.40, 131.49, 128.91, 127.35, 82.78, 71.54, 34.98, 28.85,14.63. HRMS m/e 222.0718 (M⁺) calcd for C₁₂ H₁₄ O₂ S 222.0715.

6.4.5. Oxidation of Keto Sulfide to Sulfoxide (21)

To a solution of the sulfide 46 (418 mg, 1.88 mmol) in dichloromethane(40 mL) is added an excess of solid sodium bicarbonate (1.0 g) and theresulting mixture cooled to -78° C. To this solution is added mCPBA (455mg, 2.63 mmol) and the reaction mixture stirred at low temperature for30 minutes. The temperature of the reaction mixture is slowly raised to0° C. and then quenched by pouring into a saturated solution of NaHCO₃.The resulting mixture is extracted with CH₂ Cl₂ (3×30 mL); the organiclayers are combined, dried over anhydrous Na₂ SO₄ and concentrated undervacuum. Flash chromatography (40% ethyl acetate-petroleumether) affordsthe sulfoxide 47 (380 mg, 85%) as a pale yellow oil. R_(f) (TLC)=0.25(40% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 500 MHz,) δ7.65-7.42 (m, 5H), 4.64 (t, J=5.93 Hz, 1H, H-1), 4.59 (q, 1H, H-5),2.80-2.40 (m, 3H, H-3, 3', 2), 1.80 (m, 1H, H-2'), 1.29 (d, J=6.93 Hz,3H, CH₃).

6.4.6.3-O-Benzyl-2,6-dideoxy-4-O-(trimethylsilyl)-α-L-galacto-pyranosyl-(1→4)-phenyl-3-O-benzyl-2,6-dideoxy-1-thio-α-L-galatopyranoside

The sulfoxide 22 (350 mg, 0.78 mmol) and nucleophile 23 (129 mg, 0.39mmol) are premixed and azeotroped together three times with distilledtoluene. Freshly distilled diethyl ether (9 mL) is added to a flamedried flask under argon and cooled to -78° C. The premixed reactants aredissolved in 6 mL of distilled dichloromethane and added to the flask.This is followed by the addition of Hunig's base (136 μL, 0.78 mmol).After stirring for 5 minutes triflic anhydride (65.6 μL, 0.19 mmol) isadded to the reaction. The reaction is followed by TLC (10% ethylacetate-petroleum ether). The reaction is warmed to -70° C. and quenchedby pouring into a saturated NaHCO₃ solution. The resulting solution isextracted with CH₂ Cl₂ (3×15 mL); the organic layers are combined anddried over anhydrous Na₂ SO₄. The solution is concentrated under vacuumand purified by flash chromatography (5% ethyl acetate-petroleum ether)to give the product dissaccharide as an oil (95 mg, 40% yield). R_(f)(TLC)=0.8 (10% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz)δ 7.45-7.10 (m, 10H), 5.68 (d, J=5.28 Hz, 1H), 5.07 (bs, 1H), 4.67 (d,J_(AB) =12.54 Hz, 1H), 4.55 (d, J_(AB) =12.54 Hz, 1H), 4.53 (s, 2H),4.23 (q, J=6.60 Hz, 1H), 4.17 (q, J=6.60 Hz, 1H), 3.85 (d, J=2.64 Hz,1H), 3.73 (m, 2H), 3.68 (d, J=1.32 Hz, 1H), 2.33 (dt, J=6.93, 12.54 Hz,1H), 2.02 (m, 1H), 1.19 (d, J=6.60 Hz, 3H), 0.89 (d, J=6.27 Hz, 3H),0.06 (s, 9H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 138.63, 138.29, 135.32,130.93, 128.90, 128.85, 128.59, 128.49, 128.20, 127.61, 127.55, 127.32,126.89, 84.39, 74.26, 73.79, 73.76, 71.14, 70.25, 68.10, 67.43, 31.72,29.54, 17.37, 17.23, 16.65, 0.63.

6.4.7.3-O-Benzyl-2,6-dideoxy-α-L-galactopyranosyl-(1→4)-phenyl-3-O-benzyl-2,6-dideoxy-1-thio-α-L-galatoranoside(48)

The product disaccharide from the previous section (70 mg, 0.11 mmol) isdissolved in freshly distilled THF. Tetrabutylammonium fluoride (500 μL,5 mmol) is added to the solution. The reaction is complete in one hour.Work up is done by pouring the reaction mixture in NaHCO₃ solution andextracting (3×15 mL) with THF. The organic layers are combined andconcentrated under vacuum. The product 48 is used without furtherpurification in the next step. R_(f) (TLC)=0.4 (25% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 7.5-7.2 (m, 10H),5.69 (d, J=5.27 Hz, 1H), 5.03 (d, J=2.97 Hz, 1H), 4.66 (d, J_(AB) =12.54HZ, 1H), 4.60 (d, J_(AB) =11.55 Hz, 1H), 4.56 (d, J_(AB) =11.22 Hz, 1H),4.50 (d, J_(AB) =11.22 Hz, 1H), 4.27 (q, J=6.93 HZ, 1H), 4.22 (q, J=6.60Hz, 1H), 3.90 (m, 1H,), 3.86 (bd, J=2.31 Hz, 1H), 3.74 (m, 1H), 3.72(bs, 1H), 2.12 (bs, OH), 1.91 (m, 2H), 1.19 (d, J=6.60 Hz, 3H), 1.01 (d,J=6.60 Hz, 3H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 138.10, 138.01, 135.22,130.86, 128.83, 128.46, 128.40, 127.79, 127.67, 127.63, 127.39, 126.88,99.15, 84.30, 74.84, 73.64, 73.21, 70.34, 70.07, 68.30, 67.97, 65.86,31.65, 29.83, 17.34, 16.62.

6.4.8. ABC trisaccharide (49)

The sulfoxide 21 (60 mg, 0.22 mmol) and the nucleophile 48 (61 mg, 0.11mmol) are premixed and azeotroped three times with distilled toluene.Freshly distilled dichloromethane (1 mL) and diethylether (5 mL) areadded to a flame dried flask and cooled under argon to -78° C. Thepremixed nucleophile and sulfoxide are dissolved in dichloromethane (3mL) and added to the cooled flask. This is followed by the addition ofHunig's base (40 μL, 0.22 mmol). After five minutes the triflicanhydride (18.5 μL, 0.11mmol) is added to the flask. The reaction isstirred for two hours between -78° and -70° C. The reaction is thenquenched by pouring into a solution of saturated NaHCO₃. The reactionmixture is extracted with CH₂ Cl₂ (3×15 mL), the organic layers combinedand dried over anhydrous Na₂ SO₄. The solution is concentrated undervacuum and purified by flash chromatography (20% ethyl acetate-petroleumether) to give the trisaccharide 49 (18 mg, 25% yield).

6.5. One Step Synthesis of the Ciclamycin 0 Trisaccharide

The sulfoxides 21 (417 mg, 1.812 mmol, 3.0 eq), 22 (541 mg, 1.2mmol, 2.0eq) and the nucleophile 23 (165 mg, 0.604 mmol, 1.0 eq) are premixed andthoroughly dried by azeotroping three times with distilled toluene. Thestarting materials are then dissolved in freshly distilled CH₂ Cl₂ (20mL) and added to a 50 mL flame dried flask under argon. To this reactionmixture is added 20 mL of freshly distilled Et₂ O followed by methylpropiolate (9.06 mmol, 15 eq). The flask is cooled to -78° C. using anacetone/dry ice bath. After 5 minutes, triflic acid (5.3 μL, 0.06 mmol,0.05 eq) is added to the reaction mixture dropwise. The reaction isfollowed by TLC (20% ethyl acetate-petroleum ether). The reactionmixture is slowly warmed to -70° C. over a period of half an hour andthen quenched by pouring it into a saturated solution of NaHCO₃ (30 mL).The resulting biphasic mixture is extracted with CH₂ Cl₂ (3×15 mL). Thecombined organic extracts are dried over anhydrous Na₂ SO₄ andconcentrated. Flash chromatography (20% ethyl acetate-petroleum ether)provides the trisaccharide 49 (99 mg 25%) as a colourless oil. R_(f)(TLC)=0.2 (20% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 500MHz,)δ 7.45-7.20 (m, 15 H), 5.67 (d, J=4.85 Hz,, 1H), 5.07 (d, J=2.64 Hz,1H), 4.98 (t, J=3.36 Hz, 1H), 4.66 (d, J=1.32 Hz, 1H), 4.19 (q, J=6.6Hz, 1H), 3.88 (m, 1H), 3.84 (bs, 1H), 3.73 (m, 1H), 2.52 (m, 1H), 2.28(m, 1H), 2.23-2.02 (m, 2H), 1.19 (d, J=6.60 Hz, 3H), 0.90 (d, J=5.27 Hz,3H), 0.88 (d, J=6.60 Hz, 3H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ211.13,139.13, 138.94, 135.74, 131.74, 131.63, 129.20, 128.69, 128.60,127.87, 127.81, 127.72, 127.69, 127.31, 99.57, 98.22, 84.80, 75.79,74.98, 74.69, 74.19, 73.40, 71.85, 70.45, 68.44, 67.59, 34.25, 31.88,31.09, 29.89, 17.48, 17.35, 14.93.

6.5.1. Ciclamycin trisaccharide (49a)

The sulfoxides 21 (190 mg, 0.8 mmol, 3.5 eq), 22a (230 mg, 0.48 mmol,2.0 eq) and the nucleophile 23a (85 mg, 0.24 mmol, 1.0 eq) are premixedand thoroughly dried by azeotroping three times with distilled toluene.The starting materials are then dissolved in freshly distilled CH₂ Cl₂(5 mL) and added to a 25 mL flame dried flask under argon. To thisreaction mixture is added 5 mL of freshly distilled Et₂ O followed bymethyl propiolate (4.8 mmol, 20 eq). The flask is cooled to -78° C.using an acetone/dry ice bath. After 5 minutes, triflic acid (5.3 μL,0.06 mmol, 0.05 eq) is added to the reaction mixture dropwise. Thereaction is followed by TLC (20% ethyl acetate-petroleum ether). Thereaction mixture is slowly warmed to -70° C. over a period of half anhour and then quenched by pouring it into a saturated solution of NaHCO₃(30 mL). The resulting biphasic mixture is extracted with CH₂ Cl₂ (3×15mL). The combined organic extracts are dried over anhydrous Na₂ SO₄ andconcentrated. Flash chromatography (30% ethyl acetate-petroleum ether)provides the trisaccharide 49a (35 mg 20%) as a colourless oil. R_(f)(TLC)=0.3 (30% ethyl acetate-petroleum ether).

6.5.2. Oxidation of the Sulfide to the Sulfoxide

The trisaccharide sulfide 49a (20 mg, 0.027 mmol) is dissolved in 15 mLof freshly distilled CH₂ Cl₂ taken in a 25 mL flask. To this solution isadded solid NaHCO₃ (500 mg) followed by mCPBA (7.8 mg, 0.045 mmol). Thereaction is followed by TLC (40% ethyl acetate-petroleum ether). Thereaction mixture is slowly warmed to -60° C. and quenched by pouringinto a saturated solution of NaHCO₃. The resulting biphasic mixture isextracted with CH₂ Cl₂ (3×10 mL); the organic layers are combined anddried over anhydrous Na₂ SO₄. The trisaccharide sulfoxide 50a(PMB-protected trisaccharide sulfoxide) is obtained as an oil (19 mg).It is used without purification for glycosylation.

6.5.3. Degradation of Marcellomycin to Obtain the Aglyconeε-Pyrromycinone

Marcellomycin, 53, an anthracycline antibiotic isolated from bohemicacid complex, has the same aglycone ε-pyrromycinone as ciclamycin (See,below). (The marcellomycin is a generous gift from Bristol-Myers SquibbCompany.) The aglycone can be obtained by removing the marcellomycintrisaccharide by acid hydrolysis. ##STR31##

The drug (75 mg) is refluxed in methanolic HCl (25 mL, 0.1N) for twohours. At the end of this Marcellomycin (75 mg, 0.175 mmol) is dissolvedin methanolic HCl (25 mL, 0.1N) and refluxed at 50° C. for two hours. Atthe end of this time the reaction mixture is concentrated under vacuumand purified by preparative thin layer chromatography (15%methanol-chloroform). The aglycone ε-pyrromy-cinone is isolated as abright red solid (21 mg, 54% yield).

6.5.4. Coupling of the Trisaccharide to the Aglycone

The PMB-protected trisaccharide sulfoxide 50a (19 mg, 25 μmol)ε-pyrromycinone (6 mg, 14.15 μmol) and stilbene (2.5 mg, 14 μmol) arepremixed and azeotroped three times with distilled toluene. Freshlydistilled ether (2 mL) is added to a 15 mL flame dried flask and cooledunder argon to -78° C. The azeotroped reactants are dissolved indistilled dichloromethane (3 mL) and added to the flask. After 10minutes, triflic anhydride (0.118 μl, 0.7 μmol) is added to the flaskand the reaction followed by TLC (15% ethyl acetate-petroleum ether).The reaction mixture is gradually warmed to -50° C. and quenched bypouring into a saturated solution of NaHCO₃. The resulting mixture isextracted with dichloromethane (3×5 mL); the organic layers are combinedand dried over anhydrous Na₂ SO₄. The solution is concentrated undervacuum and purified by flash chromatography (15% ether-methylenechloride followed by 10% methanol-methylene chloride). The product isisolated as a bright red solid (0.75 mg, 16% yield).

6.6. One-Pot Synthesis Of Homopolymers Of Different Lengths

FIG. 2 illustrates another aspect of the present invention which allowsthe synthesis of "homopolymers" of different lengths. Hence,alpha-linked homopolymers of 2-deoxy fucose with different lengthdistributions are produced by mixing in separate flasks different ratiosof the bifunctional sulfoxide 4-methoxyphenyl-3-O-benzyl-4-O-trimethylsilyl-2-deoxy-1-sulfinyl-α-L-fucopyranoside,B, with the monofunctional glycosyl acceptormethyl-3-O-benzyl-2-deoxy-α-L-fucopyranoside, A, and the base2,6-di-t-butyl-4-methylpyridine (2 equivalents relative to sulfoxide).The Table, below, indicates the reactant ratio that is used for each ofthe experiments 6.6.1-6.6.5. The mixtures are first dried thoroughly byazeotropic distillation from toluene (preferably, three times, asabove).

The mixtures are then each dissolved in 2.5-5 mL anhydrous methylenechloride and added to separate 25 mL flame dried flasks under argon. Toeach reaction mixture is added an equal volume of freshly distilleddiethyl ether. The flasks are next cooled to -78° C. using anacetone/dry ice bath. After 5 minutes, a methylene chloride solution oftriflic anhydride (1.0 equiv relative to B) is added dropwise to thereaction mixtures. The reactions are monitored by thin layerchromatography using 15% ethyl acetate/petroleum ether as the eluant.

After warming to -70° C. over a period of about half an hour, thereaction mixtures are quenched with saturated solution of NaHCO₃(approximately 30 mL each). Each of the resulting biphasic mixtures isextracted with methylene chloride (3×15 mL). The organic extracts arecombined, dried over anhydrous Na₂ SO₄ and concentrated. Flashchromatography (1:5 ethyl acetate/petroleum ether) is used to isolatethe glycosylated products from each reaction. The length distribution of"homopolymers" produced is found to vary with the ratio of A to B andalso with the total concentration of reactants in the reaction mixture,as shown in the Table III, below.

                  TABLE III                                                       ______________________________________                                        Relative Amounts Of Various "Homopolymers" Produced                           As A Function Of Molar Ratios Of Reactant And Total Concentration                   A:B      A + B!   AB    AB.sup.2                                                                           AB.sup.3                                                                           AB.sup.4                                                                            AB.sup.5                        Entry (ratio) (mmol/mL) (%)   (%)  (%)  (%)   (%)                             ______________________________________                                        6.6.1 1:1     0.088     40    --   --   --    --                              6.6.2 1:2     0.083     45    20   --   --    --                              6.6.3 1:3     0.096     60    30   8.7                                        6.6.4 1:5     0.050     50    30   8.0  1.5                                   6.6.5 1:3     0.233     30    40   17   8.4   1.7                             ______________________________________                                         AB = AB (.sup.1 H NMR, given below)                                           AB.sup.2 = AB-B (.sup.1 H NMR, given below)                                   AB.sup.3 = AB-B-B (.sup.1 H NMR, given below)                                 AB.sup.4 = AB-B-B-B (.sup.1 H NMR, given below)                               AB.sup.5 = AB-B-B-B-B (.sup.1 H NMR, given below)                        

More specifically, the results of the reaction described above, may beobtained by premixing the sulfoxide (320 mgs, 0.714 mmol, 3.0 eq),nucleophile (60 mgs, 0.238 mmol, 1.0 eq) and base (147 mgs, 0.714 mmol,3 eq). The resulting mixture is then dried by azeotropic distillationthree times from toluene. The reactants are then dissolved in 2.5 ml ofdistilled CH₂ Cl₂ and added to a 25 ml flame-dried flask under argon.The flask is then cooled to -78° C. and 2.5 ml of Et₂ O is added to thereaction. (The concentration of the sulfoxide is approximately 0.144mmol/ml.) After 10 min Tf₂ O (1.5 eq, 0.357 mmol, 60 μl) is added. Thereaction is maintained at -78° C. for 1/2hour, then allowed to warm to-70° C. and stirred at this temperature for an additional 1/2hour.

The reaction is quenched by pouring the reaction mixture into sat.NaHCO₃ solution, followed by extraction 3 times with CH₂ Cl₂, dryingover anhydrous Na₂ SO₄ and concentration. Purification is accomplishedby flash chromatography: 15% EP/PE; 20% EA/PE. The structures of thevarious oligosaccharides are supported by the proton NMR data (270 MHz,CDCl₃), in which the non-terminal B's are labeled X, X₁, or X₂, etc.:

AB disaccharide: ¹ H NMR δ 5.08, bs, 1H, H1B; 4.81, d, 1H, H1A; 4.7-4.5,4H benzyl methylenes; 4.2, q, 1H, H5B; 3.9-3.7, m, 5H, H3A, H4A, H4B,H3B, H5A; 3.3, s, 3H, methoxy; 1.8-2.1, 4H, H2A, H2B; 1.21, d, 3H, H6A(methyl); 0.9, d, 3H, H6B (methyl) ppm.

AXB trisaccharide: ¹ H NMR δ 5.06, s, 1H, H1B; 5.01, s, 1H, H1X; 4.82,d, 1H, H1A; 4.75-4.45, m, 6H benzyl methylene; 4.21, q, 2H, H5X, H5B;3.91-3.64, m. 7G, H3A, H3X, H3B, H5A, H4A, H4X, H4B; 3.29, s, 3H, A1methoxy; 2.08-1.80, d, 3H, H6A; 1.23, d, 3H, H6A (methyl) 8 0.90, 3d,6H, H6X, H6B (methyls) ppm.

AX₁ X₂ B tetrasaccharide: ¹ H NMR δ 5.27, d, 1H, H1B; 4.99, d, 2H, H1X₁,H1₂ ; 4.81, d, 1H, H1A; 4.72-4.45, m, BH benzyl methylenes; 4.2, m, 3H,H5X₁, H5₂, H5B; 3.90-3.62, m. 9H, H5A, H4A, H4X₁, H4X₂, H4B, H3A, H3X₁,H3X₂, H3B;3.29, s, 3H; 2.05-1.80, 8H, 2-deoxy, CH2A, CH2X₁, CH2X₂, CH2B;1.22, d, 3H, H6A (methyl); 1.9-1.8, 9H, H6X₁, H6X₂, H6B (methyls) ppm.

AX₁ X₂ X₃ B pentasaccharide: ¹ H NMR δ 5.06, s, 1H, H1B; 4.95, s, 3H,H1X₁, H1X₂, HX; 4.79, 1H, s, H1A; 4.7-4.4, m, 10H, benzyl methylenes;4.15, m, 4H, H5X₁, H5X₂, H5X₃, H5B; 3.3, s, 3H, Al methoxy; 1.2, 3H, d,H6A (methyl); 0.85, 12H, m, H6X₁, H6X₂, H6X₃, H6B (methyls) ppm.

AX₁ X₂ X₃ X₄ B hexasaccharide: ¹ H NMR δ 5.05, s, 1H, H1B; 4.98, s, 4H,HX₁, H1X₂, H1X₃, H1X₄ ; 4.7-4.4, 12H, benzyl methylenes; 4.2-4.1, 5H,H5X₁, H5X₂, H5X₃, H5X₄, H5B; 2.1-1.8, m, 12H, H2A, H2X₁, H2X₂, H2X₃,H2X₄, H2B; 1.2, d, 3H, H6A (methyl); 0.9-0.8, m, 15H, H6X₁, H6X₂, H6X₃,H6X₄, H6B (methyls) ppm.

6.7. Controlled Polymerization Reactions

According to another method of the present invention, a controlledpolymerization reaction is performed as follows:

The sulfoxide 22 (260 mg, 0.58 mmol), nucleophile 53 (73 mg, 0.29 mmol),and base 2, 6-di-tert-butyl-4-methyl pyridine (118 mg, 0.58 mmol) arepremixed and azeotroped three times with distilled toluene. To a 25 mLflame dried flask is added freshly distilled diethyl ether (5 mL) andcooled under argon to -78 The azeotroped reactants are dissolved indistilled dichloromethane (5 mL) and added to the flask. After 10minutes triflic anhydride (48.5 μL, 0.29 mmol) is added to the flask.The reaction is stirred at -70° C. for 45 minutes and then quenched bypouring into a saturated solution of NaHCO₃. The resulting biphasicmixture is extracted with CH₂ Cl₂ (3×15 mL); the organic layers arecombined and dried over anhydrous Na₂ SO₄. The solution is concentratedunder vacuum and purified by flash chromatography (15% ethylacetate-petroleum ether). The AB disaccharide 54 (70 mg, 45%) and theABB trisaccharide 55 (40 mg, 20%) are isolated as oils. AB dissacharide:R_(f) (TLC)=0.4 (15% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270MHz,) δ 7.4-7.2 (m, 10H), 5.06 (bs, 1H, H_(an)), 4.81 (d, J=2.97 Hz, 1H,H-1'), 4.68 (d, J_(AB) =12.53 Hz, 1H), 4.54 (s, 2H), 4.50 (d, J_(AB)=10.89 Hz, 1H), 4.16 (q, J=6.26 Hz, 1H, H-5), 3.9 (m, 1H, H-3), 3.8 (s,1H, H-4), 3.75 (q, J=5.94 Hz, 1H, H-5'), 3.70 (m, 1H, H-3'), 3.67 (d,J=1.65 Hz, 1H, H-4), 3.27 (s, 3H, OCH₃), 2.1 (m, 3H, H-2's), 1.8 (dd,J=4.62, 12.21 Hz, 1H, H-2_(ax)), 1.21 (d, J=6.6 HZ, 3H, CH₃), 0.9 (d,J=6.27 HZ, 3H, CH₃), 0.06 (s, 9H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 138.61,128.24, 128.14, 127.57, 137.32, 127.27, 127.19, 99.27, 98.89, 74.17,73.78, 72.91, 71.13, 70.22, 70.04, 67.28, 66.80, 54.67, 30.77, 29.51,17.51, 17.21, 0.58.

6.8. One-Pot Synthesis Of Glycoconjugates With Potential DNA BindingActivity

The strategy for forming multiple glycosidic linkages in solution can beused to synthesize in the same reaction several glycoconjugates withpotential DNA binding activity. Depending on the situation, theglycoconjugates can be separated and screened individually for DNAbinding activity or they can be screened as mixtures. For example, amixture of glycoconjugates, each comprised of a potential DNAintercalator and an oligosaccharide side chain, and differing one fromanother only in the length of the oligosaccharide side chain, aresynthesized as in Example 6.6, but using a 4:1 ratio of bifunctionaldonor to glycosyl acceptor. Specifically, the 2-deoxy fucosyl sulfoxidederivative B (908 mg, 1.90 mmol), the glycosyl acceptor A (294 mg. 0.48mmol), and 2,6-ditert-butyl-4-methyl pyridine (779 mg, 3.80 mmol) arecombined, dried by azeotropic distillation three times from toluene andthen dissolved in 10 mL of a 1:1 mixture of ether/methylene chloride(freshly distilled solvents). The solution is transferred to a flamedried flash under argon. The flask is cooled to -78° C. using anacetone/dry ice bath. After 5 minutes, 161.1 μL (0.96 mmol) triflicanhydride is added dropwise to the reaction mixture. The reaction isslowly warmed to -70° C. over a period of half an hour and then quenchedby pouring into a saturated solution of NaHCO₃ (30 mL). The mixture isextracted with methylene chloride (3×15 mL). The combined organicextracts are dried over anhydrous Na₂ SO₄, filtered, and the solventremoved under vacuum. The reaction is dissolved in 10mL of wet methylenechloride and treated with excess dichlorodicyanoquinone (DDQ) at roomtemperature for 1 hour to remove the p-methoxy benzyl ether protectinggroups. The solvent is then removed under vacuum and the components areseparated by flash chromatrography on silica gel.

Their relative affinities for DNA are evaluated to determine thepreferred length of the oligosaccharide side chain. Affinitychromatography can be used to identify oligosaccharides that bind toparticular receptors. For example, a mixture of compounds is passed overa column containing a solid support to which is attached a receptor ofinterest (or ligand, if the mobile phase contains a mixture of potentialreceptors). Compounds that bind to the receptor are retained on thecolumn longer than compounds that do not. Compounds can be fractionatedaccording to their affinity for the receptor.

Thus, receptors that bind carbohydrates can be attached to the solidsupport. Carbohydrate receptors may be comprised of DNA (double orsingle stranded), RNA, protein, oligosaccharides, or other molecules.Methods to attach nucleic acids, proteins, and oligosaccharides to solidsupports for use in affinity chromatography have been described. See:(a) Template Chromatography of Nucleic Acids and Proteins, Schott, H.Marcel Dekker, Inc.: New York, 1984; (b) Glycoconjugates: Composition,Structure and Function, Allen, H. J.; Kisailus, E. C., Eds. MarcelDekker: NY 1992 (and references therein). (NOTE: Retentiontimes can alsobe used to quantitate affinities for single compounds passed down theaffinity column.)

In another example, glycosyl acceptor A (FIG. 3) is premixed with2,3-p-methoxybenzyl-4-trimethylsilyl rhamnosyl sulfoxide 0 (FIG. 3) andallowed to react under conditions (e.g., temperature, solvent,concentration, donor/acceptor ratio) identical to those described above.After workup and removal of the p-methoxy benzyl protecting groups withDDQ, as above, the mixture of glycoconjugates is separated by flashchromatography on silica gel and the relative affinities of thedifferent compounds for DNA are determined. The glycoconjugates producedby the above-described methods are compared with respect to theirabilities to bind to DNA. In this way, the effects of different sugarson DNA binding affinity can be compared to identify preferred sugars.

The glycosyl acceptor A in FIG. 3 is made by glycosylating a suitablyprotected juglone derivative (obtained by the procedures of Inhoffen etal. Croatica Chem. Acta. 1957, 29, 329; Trost et al. J. Am. Chem. Soc.1977, 99, 8116; and Stork and Hagedorn J. Am. Chem. Soc. 1978, 100,3609) with compound B (FIG. 3) using Tf₂ O-Hunig's base CH₂ Cl₂ /ether(1:1) at low temperature. After a standard workup (including extraction,as described in the other Examples herein) and removal of solvent, theproduct mixture is dissolved in methylene chloride and treated withexcess tetrabutylammonium fluoride at 0° C. The solvent is then removedin vacuo and the product isolated by flash chromatography.

The general process described above may also be applied to the synthesisof mixtures of glycoconjugates containing several different sugars. Inthis case, two or more bifunctional glycosyl donors are used in thereaction. After deprotection, the resulting mixture of glycoconjugatescan be screened for DNA binding activity by passing it down a DNAaffinity column. Compounds can be fractionated according to theirretention times on the affinity column. Compounds with long retentiontimes can be isolated and identified using standard methods forstructure elucidation.

6.9. Additional Embodiments Illustrating the Catalytic GlycosylationMethod

The following examples relate to glycosylation methods mediated bycatalytic amounts of sulfonic acid, including reactions involvingsilylated glycosyl acceptors.

6.9.1. 1,2,3,4-Tetra-O-acetyl-6-)-(trimethylsilyl)-D-glucopyranose (2b)

The following procedure is typical for all silylations of thenucleophile. The alcohol 2a (600 mg, 1.85 mmol, 1.0 eq) and triethylamine (775 μL, 3.43 mmol, 3.0 eq) are dissolved in distilled methylenechloride and cooled to -78° C. under argon. Trimethyl silyl triflate(394 μL, 2.03 mmol, 1.1 eq) is added to the reaction mixture. Thereaction is followed by TLC. After 30 minutes the reaction is quenchedby pouring into a saturated solution of NaHCO₃. The resulting biphasicmixture is extracted with CH₂ Cl₂ (3×25 mL). The organic layers arecombined and dried over anhydrous Na₂ SO₄. Purification by flashchromatography affords the silyl ether 2b (700 mg, 90%) as a whitesolid. R_(f) (TLC)=0.6 (40% ethyl acetate-petroleum ether). ¹ H NMR (270MHz, CDCl₃) δ 5.69 (d, J=8.25 Hz, IH, H-1), 5.25 (t, J=9.24 Hz, IH,H-3), 5.15 (t, J=9.24 Hz, 1H, H-4), 5.12 (t, J=8.24, 9.25 Hz, IH, H-2),3.8-3.7 (m, 3H, H-5, H-6,6'), 2.1-2 (m, 12H, OAc), 0.5 (s, 9H, TMS).

6.9.2. α,α-1,1-Dimer of Perbenzylated Glucose (5)

The perbenzylated glucose 1,1-dimer is identified, as follows: R_(f)(TLC)=0.8 (25% ethyl acetate-petroleum ether). ¹ H NMR (270 MHz, CDCl₃)δ 7.4-7., 1 (m, 40H), 5.19 (d, J=3.30 Hz, 1H), 5.16 (d, J_(AB) =11.55Hz, 1H), 5.02 (d, J_(AB) =10.89 Hz, 1H), 4.97 (d, J_(AB) =12.21 Hz, 1H),4.90 (d, J_(AB) =10.89 Hz, 1H), 4.89 (d, J_(AB) =10.89 Hz, 1H), 4.83 (d,J_(AB) =10.89 Hz, 1H), 4.82 (d, J=3.30 Hz, 1Hah), 4.81 (d, J_(AB) =11.54Hz, 1H), 4.75 (d, J_(AB) =12.21 Hz, 1H), 4.69 (d, J_(AB) =12.21 Hz, 1H),4.59 (bs, 2H), 4.55 (d, J_(AB) =11.88 Hz, 1H), 4.52 (d, J_(AB) =10.88Hz, 1H), 4.49 (bs, 2H), 4.34 (d, J_(AB) =12.21 Hz, 1H), 4.21 (m, 1H),4.15 (t, J=9.24, 9.47 Hz, 1H), 3.82 (t, J=9.24, 9.9, Hz, 1H), 3.7-3.45(m, 7H).

6.9.3. Synthesis of3,4,6-Tri-O-benzyl-2-deoxy-D-gluco-pyranosyl-(1→6)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranose(7)

The following procedure is typical of all glycosylation reactions rununder acid catalyzed conditions using TfOH: The sulfoxide 6 (140 mg,0.258 mmol, 1.5 eq) and the nucleophile 2 (69 mg, 0.172 mmol, 1.0 eq.)are premixed and thoroughly dried by "azetroping "3 times with distilledtoluene. The starting materials are then dissolved in 8 mL of freshlydistilled CH₂ Cl₂ and added to a 25 mL flame dried flask under argon.The flask is cooled to -78° C. using an acetone-dry ice bath. Methylpropiolate (230 μL, 2.58 mmol, 15 eq) is added to this solution as asulfenic acid scavenger. After the solution is stirred at -78° C. for 2minutes, triflic acid (1.1 μL, 0.0129 mmol, 0.05 eq) is added. Thereaction is followed by TLC (25% ethyl acetate-petroleum ether). Thereaction mixture is slowly warmed to -30° C. over a period of 1 hour,and then quenched by pouring it into a saturated solution of NaHCO₃ (25mL). The resulting mixture is extracted with CH₂ Cl₂ (3×15 mL). Theorganic extracts are combined, dried with anhydrous Na₂ SO₄, andconcentrated. Flash chromatography (25% ethyl acetate-petroleum ether)provides the disaccharide 7 (116 mg, 88%) as a white solid. R_(F)(TLC)=0.3 (25% ethyl acetate-petroleum ether). ¹ H NMR (270 MHz, CDCl₃)δ 7.4-7.2 (m, 15H), 5.75 (d, J=8.25 Hz, 1H, H-a), 5.32 (t, J=9.24 Hz,1H, H-c), 5.21 (t, J=9.57 Hz, 1H, H-d), 5.20 (t, J=8.25, 9.24 Hz, 1H,H-b), 4.99 (d, J=2.64 Hz, 1H, H-1), 4.95 (d, JAB=11.21 Hz, IH), 4.71(bs, 2H), 4.66 (d, J_(AB) =11.87 Hz, 1H), 4.59 (d, J_(AB) =11.22 Hz,1H), 4.55 (d, J_(AB) =11.88 Hz, 1H), 4.10 (m, 1H, H-e), 3.85-3.50 (m,7H), 2.36 (dd, J=4.94, 12.86 Hz, 1H), 2.15 (s, 3H), 2.10 (s, 3H), 2.05(s, 3H), 1.95 (s, 3H), 1.70 (m, 1H).

6.9.4.Methyl-6-deoxy-3,4-isopropylidene-2-O-(trimethylsilyl)-β-D-galactopyranoside(8)

The compound 8 is synthesized from D-fucose by the following three-stepsequence: (i) MeOH, H⁺ ; (ii) acetone, H₃ PO₄ (cat); (iii) TMSOTf, Et₃N, CH₂ Cl₂, -78° C. Rf (TLC) =0.5 (15% ethyl acetate-petroleum ether). ¹H NMR (CDCl₃, 270 MHz) δ 4.03 (d, J=8.24 Hz, 1H, H-1), 3.98 (dd, J=5.28,7.26 Hz, 1H), 3.97 (dd, J=5.40, 1.98 Hz, 1H), 3.84 (dq, J=1.98, 6.6 Hz,1H, H-5), 3.49 (s, 3H, OCH₃), 3.47 (dd, J=7.59, 5.49 Hz, 1H), 1.48 (s,3H, CH₃), 1.38 (d, J=6.6 Hz, 3H, CH₃) 1.31 (s, 3H, CH₃), 0.5 (s, 9H,TMS). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 109.38, 103.70, 80.52, 76.48, 74.45,68.58, 56.64, 28.02, 26.34, 16.49, 0.31.

6.9.5. 3,4,6-Tri-O-benzyl-2 -deoxy-D-glucopyranosyl-α-(1→2)-methyl-6-deoxy-3,4-isopropylidene-β-D-galactopyranoside (9)

R_(f) (TLC)=0.5 (25% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270MHz) α anomer δ 7.4-7.15 (m, 15H), 5.30 (d, J=2.97 Hz, 1H), 4.86 (d,J_(AB) =10.89 Hz, 1H), 4.64 (d, J=1.65 Hz, 2H), 4.62 (d, J_(AB) =12.20Hz, 1H), 4.53 (d, J_(AB) =10.89 Hz, 1H), 4.45 (d, J_(AB) =12.20 Hz, 1H),4.06 (d, J=8.25 Hz, 1H), 3.70 (t, J=9.56 Hz, 1H), 3.46 (s, OCH₃), 2.02(ddd, J=0.99, 4.95, 12.87 Hz, 1H), 1.65 (dt, J=3.62, 12.87 Hz, 1H), 1.40(s, CH₃), 1.36 (d, J=6.6 Hz, 3H), 1.24 (s, 3H). ¹³ C NMR (CDCl₃, 67.9MHz) δ 138.91, 138.83, 138.32, 128.28, 128.23, 127.92, 127.79, 127.45,127.42, 127.38, 109.35, 103.95, 97.45, 78.39, 78.20, 77.70, 76.31,76.12, 74.95, 73.45, 71.73, 70.53, 68.73, 68.48, 56.58, 35.47, 28.08,26.34, 16.49.

6.9.6.Phenyl-4-O-acetyl-2,3,6-trideoxy-1-sulfinyl-α-L-erythro-hexopyranoside(10)

Compound 10 is synthesized from L-rhamnal in 3 steps. a) H₂ (1 arm),Pd(OH)₂ /C, C₆ H₆ ; b) BF₃.OEt₂, thiophenol, CH₂ Cl₂, -78° C. to -60°C.; and c) mCPBA, CH₂ CI₂, -78° C. to -60° C. R_(f) (TLC)=0.4 (25% ethylacetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) β anomer (sulfide) δ7.5-7.2 (m, 5H), 5.54 (d, J=4.95 Hz, H-4), 4.81 (dd, J=1.98, 12.1 Hz,1H, H-i), 4.6 (m, 2H, H-3, 3'), 4.3 (m, 1H, H-5), 2.2 (m, 2H, H₂,2 '),2.1 (s, 3H, OAc), 1.19 (d, J=5.94 Hz, 3H, CH₃). ¹³ C NMR (CDCl₃, 67.9MHz) β anomer (sulfide) δ 169.80, 130.98, 130.73, 128.58, 128.49, 83.79,67.15, 29.95, 25.34, 20.82, 18.00, 17.44.

6.9.7. 4-O-Acetyl-2,3,6-trideoxy-L-erythro-hexopyranosyl-α-(1→6)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranose (11)

R_(f) (TLC)=0.3 (25% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270MHz) α anomer δ 5.63 (d, J=7.92 Hz, 1H), 5.20 (t, J=9.24 Hz, 1H), 5.19(t, J=9.24, 9.57 Hz, 1H), 5.09 (d, J=7.91 Hz, 1H), 5.03 (t, J=9.57 Hz,1H), 4.61 (dd, J=1.98, 8.58 Hz, 1H), 4.33 (ddd, J=4.61, 10.23, 10.23 Hz,1H), 3.94 (dd, J=3.96, 11.22 Hz, 1H), 3.71 (m, 1H), 3.56 (dd, J=2.96,11.22 Hz, 1H), 2.05 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H),1.95 (s, 3H), 1.11 (d, J=6.59 Hz, 3H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ170.19, 170.12, 169.18, 169.15, 168.95, 100.85, 91.79, 73.03, 72.96,70.34, 68.60, 66.01, 29.54, 26.88, 21.07, 20.76, 20.64, 20.54, 20.50,18.01, 17.72.

6.9.8. Phenyl-3,4-bis-O-(4-methoxybenzoyl)-1-sulfinyl-D-digltoxose (13)

Compound 13 is synthesized from digitoxose by the following three-stepsequence: (i) pOMeC₆ H₄ COCI, pyridine; (ii) thiophenol, Et₂ O.BF₃, CH₂Cl₂ ; (iii) mCPBA. R_(f) (TLC)=0.2 (40% ethyl acetate-petroleum ether).¹ H NMR (CDCl₃, 270 MHz) α anomer (sulfoxide) δ 8.09 (d, J=8.91 Hz, 2H),7.87 (d, J=9.24 Hz, 2H), 7.70 (m, 2H), 7.55 (m, 3H), 6.94 (d, J=8.90 Hz,2H), 6.85 (d, J=8.91 Hz, 2H), 5.84 (q, J=3.31 Hz, 1H), 5.10 (dd,J:=2.63, 2.97, 1H), 5.04 (m, 1H), 4.49 (dd, J=1.64, 5.13 Hz, 1H), 3.83(s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 1.28 (d, J=5.95 Hz, 3H).

6.9.9.Phenyl-3,4-O-(4-methoxybenzoyl)-β-D-digitoxosyl-(O)-N-hydroxyethylcarbamate (14)

R_(f) (TLC)=0.45 (cc anomer, 40% ethyl acetate-petroleum ether). ¹ H NMR(CDCl₃, 270 MHz) α anomer δ 8.03 (d, J=8.91 Hz, 2H), 7.79 (d, J=9.24 Hz,2H), 6.88 (d, J=9.24 Hz, 2H), 6.77 (d, J=9.01 Hz, 2H), 5.60 (q, 1H),5.12 (d, J=4.62 Hz, 1H), 4.95 (dd, J=2.97, 10.23 Hz, 1H), 4.78 (m, 1H),4.X8 (q, 2H), 4.17 (m, 1H), 3.84 (s, OCH₃), 3.79 (s, OCH₃), 2.37 (dd,J=3.3, 15.51 Hz, 1H), 2.19 (m, 1H), 1.26 (t, J=6.93 Hz, 3H),, 1.25 (d,J=6.27 Hz, 3H). ¹³ C NMR (CDCl₃, 67.9 MHz) δ 165.48, 165.17, 163.53,163.45, 157.42, 131.97, 131.75, 122.65, 121.92, 113.62, 113.59, 100.69,100.61, 72.21, 65.90, 63.65, 62.08, 55.40, 32.04, 17.53, 14.44 R_(f)(TLC)=0.5 (β anomer, 40% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃,270 MHz) β anomer δ 7.92 (d, J=8.91 Hz, 2H), 7.78 (d, J=8.91 Hz, 2H),6.89 (d, J=8.91 Hz,, 2H), 6.77 (d, J=9.24 Hz, 2H), 5.74 (q, 1H), 5.23(dd, J=2.31, 9.23 Hz, 1H), 4.93 (dd, J=2.97, 9.23 Hz, 1H), 4.23 (q, 1H),4.16 (q, 2H), 4.17 (m, 1H), 3.85 (s, OCH₃), 3.79 (s, OCH₃), 2.36 (m,1H), 2.04 (m, 1H), 1.29 (d, J=6.27 Hz, 3H), 1.23 (t, J=6.93 Hz, 3H). ¹³C NMR (CDCl₃, 67.9 MHz) δ 165.10, 164.91, 163.61, 163.55, 131.85,122.18, 121.83, 113.74, 113.59, 101.77, 72.31, 69.21, 67.41, 62.19,60.34, 55.44, 55.38, 33.29, 18.01, 14.38.

6.9.10. Phenyl-2,3-O-benzoyl-6-deoxy-1-thio-β-L-galactopyranoside (15)

The compound 15 is synthesized from L-fucose by the following four-stepsequence: (i) Ac₂ O, pyridine; (ii) thiophenol, Et₂ O.BF₃ ; (iii) NaOMe,CH₃ OH, amberlite H⁺ resin; (iv) C₆ H₅ COCl, DMAP. R_(f) (TLC)=0.6 (40%ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270 MHz) δ 8.0-7.9 (m,4H), 7.55-7.2 (m, 1H), 5.64 (t, J=9.89 Hz, 1H), 5.27 (dd, J=2.97, 9.9Hz, 1H),, 4.86 (d, J=9.9 Hz, 1H), 4.09 (d, J=2.97 Hz, 1H), 3.87 (q,J=6.27 Hz, 1H), 1.40 (d, J=6.27 Hz, 3H).

6.9.11. Phenyl-3,4-O-acetyl-2,6-dideoxy-1-sulfinyl-L-galactopyranoside(16)

Compound 16 is prepared from 1,3,4-tri-O-acetyl-2-deoxy-α-L-fucose bythe following two-step sequence: (i) thiophenol, Et₂ O.BF₃ ; and (ii)mCPBA. R_(f) (TLC for a sulfide) =0.3 (15% ethyl acetate-petroleumether). ¹ H NMR (CDCl₃, 270 MHz) a sulfide δ 7.5-7.2 (m, 5H), 5.73 (d,J=5.61 Hz, 1H, H-1), 5.29 (m, 1H, H-3), 5.23 (bs, 1H, H-3), 4.56 (q,J=6.6 Hz, 1H, H-5), 2.49 (dt, J=5.94, 12.87 Hz, 1H, H-2_(ax)), 2.38 (s,3H, OAc), 2.06 (m, 1H, H-2_(eq)), 1.99 (s, 3H, OAc), 1.13 (d, J=6.6 Hz),3H, CH₃). ¹³ C NMR a sulfide (CDCl₃, 67.9 MHz) δ 170.51, 169.87, 159.54,134.33, 124.54, 114.54, 84.58, 69.69, 67.18, 65.51, 55.22, 20.79, 20.60,16.34. R_(f) (TLC for a sulfoxide)=0.25 (30% ethyl acetatepetroleumether). ¹ H NMR α anomer sulfoxide (CDCl₃, 270 MHz) δ 7.6-7.3 (m, 5H),5.43 (m, 1H), 5.26 (bd, J=2.64 Hz, 1H), 4.52 (d, J=5.61 Hz, 1H), 4.32(q, J=6.6 Hz, 1H), 2.47 (dd, J=5.28, 14.18 Hz, 1H), 2.14 (dt, J=5.61,14.19 Hz, 1H), 2.10 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.12 (d, J=6.26 Hz,3H).

6.9.12.3,4-O-Acetyl-2,6-dideoxy-L-galactopyranosyl-α-(1→4)-phenyl-3,4-O-benzoyl-6-deoxy-1-thio-β-L-galactopyranoside(17)

R_(f) (TLC)=0.4 (30% ethyl acetate-petroleum ether). ¹ H NMR (CDCl₃, 270MHz) δ 7.9 (m, 4H), 7.55-7.25 (m, 1H), 5.55 (t, J=10.23 Hz, 1H), 5.20(dd, J=2.96, 10.22 Hz, 1H), 5.12 (m, 1H), 5.02 (bd, J=l. 98 Hz, 1H),4.94 (bs, 1H), 4.84 (d, J=9.9 Hz, 1H), 4.15 (d, J=2.97 Hz, 1H), 3.96 (q,J=6.27 Hz, 1H), 3.86 (q, J=6.27 Hz, 1H), 2.04 (s, 3H), 2.0 (s, 3H), 1.94(m, 2H), 1.35 (d, J=6.27 Hz, 3H), 0.33 (d, J=6.27 Hz, 3H). ¹³ C NMR(CDCl₃, 67.9 MHz) δ 170.44, 169.62, 166.05, 164.95, 133.51, 133.36,133.01, 131.49, 129.78, 129.62, 129.12, 128.65, 128.44, 128.27, 128.18,99.49, 85.42, 76.86, 75.27, 74.68, 69.56, 67.47, 66.50, 65.27, 29.70,20.85, 20.56, 17.32, 15.77.

6.10. Synthesis of a β-Linked Disaccharide on the Solid Phase

To a 10 mL solution of DMF containing 0.338 g (0.350 mmol) of2,3,4-tribenzyl-6-tritylgalactose-1-p-hydroxy phenythioglycoside cesiumacetate (X, FIG. 6) is added 0.356 g (0.385 mmol Cl equiv., 1,1 equiv.)of Merrifield resin (BACHEM Bioscience). This mixture is agitated with awrist action shaker for 24 h under argon atmosphere at 75° C. At thistime, the polymer is poured into a tared coarse-fritted Gooch funnel andwashed repeatedly with methanol and methylene chloride. The funnel isthen dried for 4 h in a lyophilizer jar at 20 milliTorr. A mass changeof 0.244 g is recorded, which is calculated to be 85% chemical yieldwith respect to the cesium salt.

The polymer in the coarse-fritted Gooch funnel is then treated by vacuumfiltration at moderate flow rate with 40 mL of 10% trifluoroacetic acid(TFA) in methylene chloride until no yellow color is apparent in thefiltrate. (The TFA is used to remove the trityl protecting groups.) Thepolymer is next washed repeatedly with methanol and methylene chloride.The funnel, together with the resin-linked nucleophile, is then driedfor 4 h in a lyophilizer jar at 20 milliTorr before "massing". A masschange of 0.065 g is subsequently measured, which is calculated to be83% chemical yield with respect to the cesium salt. The concentration ofthe resin-linked nucleophile (resin-X, FIG. 6) is then calculated to be0,544 mmol/g.

200 mg (i.e., 0.11 mmol) of derivatized resin is lyophilized overnightin the reaction vessel and then purged for 1 hour with argon. The resinis then suspended in 5 mL CH₂ Cl₂. 4 equivalents (0.44 mmol) of2-pivaloyl-3,4-benzyl-6-p-methoxy benzyl galactosyl phenyl sulfoxide (Y,FIG. 6) and 6 equivalents (0.66 mmol) 2,6-di-t-butyl-4-methyl pyridineare dissolved in 5 mL methylene chloride and added by canula to thereactor vessel. The mixture is agitated gently by argon flow for 30minutes at room temperature and then the reactor vessel is immersed in acold bath and allowed to cool to -60° C. 2 equivalents (0.22 mmol) oftriflic anhydride diluted one hundred fold (v/v) in methylene chlorideare added slowly (over 15 minutes) to the reaction vessel. The resultingreaction mixture is gently agitated for 1 hour.

After the reaction is completed, as indicated by the Hg(II) hydrolysismethod and TLC analysis, the solvent and unbound reagents are thendrained from the reactor vessel, and the resin mixture is rinsedrepeatedly with methanol followed by methylene chloride. Subsequently,the resin mixture is suspended in 15 mL of methylene chloride and thentreated with excess Hg(OCOCF₃)₂ for 8 hours to cleave the glycosidiclinkage to the resin. (Note: only 5 minutes is required to removesufficient product from the resin to monitor the reaction by TLCanalysis.) The solvent is allowed to drain from the resin. Additionalsolvent is then used to rinse the resin. The filtrates are thencombined, extracted three times with water and concentrated byevaporation. The desired β-linked disaccharide is obtained by flashchromatography on silica gel. No α-linked disaccharide is isolated fromthe reaction.

Thiosugar X in FIG. 6 is prepared from the readily available1,6-anhydroglucose by treatment with benzyl bromide followed by acidichydrolysis (H₂ SO₄ -THF-H₂ O), tritylation (trityl chloride-pyridine) ofthe more reactive C6 primary alcohol, and treatment of the resultinglactol with disulfide XX and tri-n-butylphosphine (i.e., standardprocedure for making thiophenyl glycosides from lactols). The disulfideXX is produced by reacting the disulfide of the readily available4-hydroxythiophenol with α-bromo-methyl acetate. ##STR32##

Sulfoxide Y in FIG. 6 is prepared from readily availablepenta-acetylated galactose using the following sequence of reagents: (1)BF₃ /etherate-ethiophenol; (2) hydroxide; (3) acetone-H⁺ ; (4) p-methoxybenzyl bromide-sodium hydride; (5) pivaloyl chloride; (6) mCPBA. Eachstep is well known in the art and the reactions are carried out underthe standard conditions. (See, list of "Standard References" below inSection 6.15 the disclosures of which are incorporated by referenceherein.).

The disaccharide produced from the reaction of X and Y is subjected tomethanolysis (to remove it from the resin) and is characterized by ¹ HNMR spectroscope. The relevant data include: (CDCl₃) 5.58 ppm (d, J=5.3Hz, H1 of thiosugar), 5.47 ppm (dd, J=7.9, 10.2 Hz, H2 of C2pivaloylated sugar), 4.45 ppm (d, partially overlapped, H1 of C2pivaloylated sugar).

6.11. Synthesis of an α-Linked Disaccharide on the Solid Phase

The sodium salt of a glycosyl acceptor (X, FIG. 7) is attached to theMerrifield resin by the standard method (DMF, 80° C., 24 h). Followingthe coupling and rinsing (as described in Example 6.10), the resin islyophilized and weighed. Loading is calculated at 0.52 mmol/g from themass gain.

200 mg (i.e., 0.1mmol) of derivatized resin is lyophilized overnight inthe reaction vessel (FIG. 7) and then purged for 1 hour with argon. Theresin is then suspended in 5 mL methylene chloride. 4 equivalents (0.4mmol) of perbenzylated fucosyl sulfoxide Y and 6 equivalents of2,6-di-t-butyl-4-methyl pyridine are dissolved in 5 mL methylenechloride and added by syringe to the reactor vessel. The mixture isagitated gently by argon flow for 30 minutes at room temperature andthen the reactor vessel is immersed in a cold bath and allowed to coolto -60° C. 2 equivalents (0.22 mmol) of triflic anhydride diluted onehundred fold in methylene chloride are added slowly by syringe (over 15minutes) to the reaction vessel. The reaction is gently agitated for 1hour. The solvent and unbound reagents are then drained from the reactorvessel, and the resin mixture is rinsed repeatedly with methanolfollowed by methylene chloride. Subsequently the resin mixture issuspended in 15 mL of methylene chloride and then treated with excessHg(OCOCF₃)₂ for 8 hours to cleave the glycosidic linkage to the resin(only 5 minutes is required to remove sufficient product from the resinto monitor the reaction by TLC analysis). The solvent is allowed todrain from the resin as before. The resin is then rinsed with additionalsolvent and the filtrates combined, extracted three times with water,and concentrated by evaporation. Flash chromatography on silica gelgives only the desired α-linked disaccharide.

Thiosugar X in FIG. 7 is prepared from the readily availablecorresponding glucosamine by treatment with the following reagents: (1)phthalic anhydride; (2) acetic anhydride; (3) tetrachlorotin-4-hydroxythiophenol; (4) hydroxide; (5) benzaldehyde-H⁺ ; (6) NaH, underconditions that are standard in the art. (See, Section 6.15, below).

Sulfoxide Y in FIG. 7 is made from peracetylated fucose by treating thestarting material sequentially with BF₃ /etherate-thiophenol, followedby hydroxide, followed by benzyl bromide, and then with mCPBA. All thesesteps are standard and well known in the art.

The disaccharide produced from the reaction of X and Y followingtreatment with Hg(OCOCF₃)₂ (to remove it from the resin) ischaracterized by ¹ H NMR. Relevant data: CDCl₃) 5.6 ppm (apparent t,J=7.6 Hz, H1 of C2 phthalimido sugar), 3.35 ppm (d, J=7.6 Hz, lactol OH,i.e., of phthalimido sugar after hydrolytic removal from resin w/Hg(II),4.9 ppm (d, J=2.8 Hz, H1 of fucose derivative).

6.12. Solid Phase Synthesis Of Lewis X Trisaccharide

The sodium salt of a glycosyl acceptor (X, FIG. 8) is attached to theMerrifield resin using the standard method (DMF, 80° C., 24 h). Afterusing the general linking procedure described in detail in Example 6.10,the anhydrous resin is suspended in 5 mL methylene chloride. 4 eq.2,3,4,6-pivaloylated galactosyl sulfoxide Y and 6 eq. base (as above) isdissolved in 5 mL methylene chloride. The reagent solution is then addedto the resin, and the reaction mixture is cooled to -60° C. 2equivalents of triflic anhydride diluted one hundred fold (v/v) inmethylene chloride are then added.

After 30 minutes, the resin is "drained" and rinsed repeatedly withmethylene chloride and methanol. The resin is then suspended in 5 mLmethylene chloride and cooled to 0° C. 5 mL of a 1:2 solution oftrifluoroacetic acid/methylene chloride is then added, and the resin isagitated gently for 5 hours. The resin is then "drained" and rinsedrepeatedly with methylene chloride and methanol. Following a final rinsewith anhydrous methylene chloride, the resin is suspended in 5 mL ofanhydrous methylene chloride. 4 equivalents of 2,3,4-triethylsilylfucosyl sulfoxide Z and 6 equivalents of hindered base (i.e., that usedabove) are dissolved in 5 mL methylene chloride and added to the resin.The reaction mixture is cooled to -60° C. 2 equivalents of triflicanhydride are diluted 100 fold in methylene chloride and added slowly bysyringe to the reaction. After agitating gently for 30 minutes, theresin is "drained" and rinsed. The trisaccharide is then removed andisolated from the resin using Hg(OCOCF₃)₂, as described above in Example6.10.

Thiosugar X in FIG. 8, is prepared from the readily availableglucosamine by treatment with (1) phthalic anhydride; (2) aceticanhydride; (3) tetrachlorotin-4-hydroxy thiophenol; (4) benzyl bromide;(5) hydroxide; (6) benzaldehyde-H⁺ ; (7) chloromethyl methyl ether (MOMchloride); (8) H₂ O/H⁺ ; (9) pivalyoyl chloride; (10) hydrogenation,Pd(OH)₂ ; (11) NaH. Again, all steps are standard including theconditions for the deprotection of the benzyl protecting group on the4-hydroxy thiophenyl glycoside, which conditions are typical fordebenzylation. In the debenzylation step, no cleavage of the sugar tosulfur bond is observed.

Sulfoxide Y in FIG. 8 is prepared by treating perpivaloylated galactosewith BF₃ /etherate-thiophenol, followed by mCPBA.

Sulfoxide Z in FIG. 8 is prepared by treating peracetylated fucose withBF₃ /etherate-thiophenol, followed sequentially by hydroxide,triethylsilyl chloride, and mCPBA.

Similarly, other Lewis blood group sugars are synthesized readily,including, but not limited to, Lewis A and Lewis B.

6.13. Additional Experiments Conducted in the Solid Phase

Referring to FIG. 16, in particular, cesium fluoride (157.2 mg, 1.03mmol, 1.1 eq.) and Merrifield resin (1 mol. eq. Cl per gram) (1.0975 g,1.10 mol, 1.1 eq.) are added to a solution of freshly prepared 1 (803.5mg, 0.94 mmol, 1.0 eq.) in DMF (20 mL). The resulting suspension isshaken mechanically under an argon atmosphere at 60° C. for 24 h. Thesugar-derivatized resin is then isolated by vacuum filtration and washedwith DMF (3×10 mL), methanol (3×10 mL) and CH₂ Cl₂ (5×10 mL) and thendried under vacuum overnight to give resin 2 (1.4738 g, calculated basedon mass gain, or 0.34 mmol/g). IR (KBr disc) 1734 cm⁻¹ (υ, C═O).

Resin 2 is then washed with 10% trifluoroacetic acid in CH₂ Cl₂ (20 mL)to remove the protecting group. Subsequently, the deprotected resin 3 iswashed with CH₂ Cl₂ (10×10 mL) and dried under vacuum overnight. IR (KBrdisc) 1734 cm⁻¹ (υ, C═O), 3430 cm⁻¹ (br, u, O--H).

To obtain resin 5, 3 (95.4 mg) is added to a solution of the sulfoxide 4(122.1 mg, 0.156 mmol, 1.0 eq.) and 2,6-di-tert-butyl-4-methyl-pyridine(98.2 mg, 0.468 mmol, 3.0 eq.) in CH₂ Cl₂ (7.0 mL) and the suspensioncooled to -78° C. After 10 mins, triflic anhydride (13.1 μL, 0.078 mmol,0.5 eq.) in CH₂ Cl₂ (5.0 mL) is added dropwise over a period of 20 mins.Ten minutes after the addition is complete, the reaction is warmed to-60° C. over a period of 20 mins. The reaction is quenched by theaddition of saturated NaHCO₃. The resin is collected by suctionfiltration and washed with methanol (2×10 mL), water (1×10 mL), methanol(1×10 mL), and then CH₂ Cl₂ (10×10 mL). The product resin 5 is thendried under vacuum overnight, after which the glycosylation is repeated.

The product resin 6 is obtained as follows: resin 5 (112.7 mg) is washedwith 10% trifluoroacetic acid in CH₂ Cl₂ (20 mL). It is then washed withCH₂ Cl₂ (10×10 mL) and dried under vacuum overnight.

Then resin 7 is obtained from resin 6 as follows: resin 6 (110.7 mg) isadded to a solution of sulfoxide 4 (123.5 mg, 0.158 mmol, 1.0 eq. ) and2,6-di-tert-butyl-4-methyl-pyridine (99.3 mg, 0.474 mmol, 3.0 eq.) inCH₂ Cl₂ (7.0 mL) and the suspension cooled to -78° C. After 10 mins,triflic anhydride (13.3 μL, 0.079 mmol, 0.5 eq.) in CH₂ Cl₂ (5.0 mL) isadded dropwise over a period of 20 mins. Ten minutes after the additionis complete, the reaction is warmed to -60° C. over a period of 20 mins.The reaction is quenched by the addition of saturated NaHCO₃. The resinis isolated by filtration and washed with methanol (2×10 mL), water(1×10 mL), methanol (1×10 mL), and then CH₂ Cl₂ (10×10 mL). The productresin 7 is dried under vacuum overnight, after which the glycosylationis repeated.

Finally, compound 8 is obtained from resin 7 as follows: resin 7 (113.6mg) is washed with 10% trifluoroacetic acid in CH₂ Cl₂ (20 mL). It isthen washed with CH₂ Cl₂ (10×10 mL) and suspended in pyridine (10 mL).Acetic anhydride (183.7 μL, 3..932 mmol, 1.0 eq.) and4-dimethylaminopyridine (4.7 mg, 0.039 mmol, 0.02 eq.) are added to thesuspension under argon. The mixture is stirred overnight and thenquenched by the addition of methanol (200 μL). The resin is isolated byfiltration and washed with methanol (2×10 mL) and CH₂ Cl₂ (10×10 mL).The free compound 8 is released from the resin by treatment with bis(trifluoroacetato)mercury(II) in wet methylene chloride, as describedbelow, for example, for the synthesis of disaccharide 13. (compound 8) ¹H NMR (500 MHz, CDCl₃) δ 4.42 (d, J =8.06 Hz, H₁ "), 4.49 (d, J=8.06 Hz,H₁ '), 6.27 (d, J =3.67 Hz, H₁). ¹³ C NMR (69 MHz, CDCl₃) δ 91.6, 101.0,101.3 (anomeric carbon atoms).

6.14. Solid Phase Synthesis of Disaccharides 13 and 16

According to the scheme of FIG. 17, the Pht-protectedN-acetylglucosamine residue 9 is attached to a solid support, asfollows: Merrifield resin (1.4075 g, 1% cross-linked, chloromethylatedstyrene/divinylbenzene copolymer, approximately 1 mmol Cl/g, Aldrich) isadded to a solution of freshly prepared 9 (1.42 g, 2.693 mmol) in DMF(20 mL). The suspension is shaken mechanically at 60° C. under argon for24 h. The sugar-derivatized resin is isolated by filtration, washed withDMF (5×10 mL), methanol (3×10 mL) and CH₂ Cl₂ (10×10 mL), and driedunder vacuum overnight to give 1.7466 g of resin-attached 10. The levelof loading is calculated to be 0.414 mmol/g (based on mass gain). IR(KBr) 1670, 1716 cm⁻¹ (υ, C═O); 3470 cm⁻¹ (υ, C═O).

Resin 10 is coupled to sulfoxide 11, a fucose unit, to give 12(α-glycosidic linkage), as follows: Compound 10 (218.4 mg) is added to asolution of sulfoxide 11 (204.1 mg, 0.3766 mmol, 1.0 eq.) and2,6-di-tert-butylpyridine (236.7 mg, 1.1297 mmol, 3.0 eq.) in CH₂ Cl₂ (7mL). The suspension is cooled to -60° C. and, after 10 mins, a solutionof triflic anhydride (31.7 μL, 0.1883 mmol, 0.5 eq.) in CH₂ Cl₂ (5 mL)is added dropwise over a period of 20 min. Ten minutes after theaddition is complete, the reaction is slowly warmed to -30° C. and,after 30 mins, quenched by the addition of saturated aqueous sodiumbicarbonate. The resin is isolated by filtration, washed with methanol(2×10 mL), water (2×10 mL), methanol (10 mL) and CH₂ Cl₂ (10×10 mL), anddried under vacuum overnight to give 12.

The final product 13 is obtained as follows: Hg(OCOCF₃)₂ (120.5 mg0.2824 mmol) and water (a few drops) are added to a suspension of 12(222.0 mg) in CH₂ Cl₂ (20 mL). After 5 h the mixture is filtered throughcotton. The filtrate is washed with saturated sodium bicarbonate, driedover Na₂ SO₄, filtered, and concentrated in vacuo. The residue ispurified by flash chromatography (35% EtOAC-pet. ether) to give 33.9 mgof 13 (59% yield) as a colorless solid. This solid could be purifiedfurther by RP-HPLC (C-18 column, 60 to 98% MeCN in water over 30 mins),R_(T) 5.7 mins. ¹ H NMR (500 MHz, CDCl₃) δ 4.81 (d, J =3.66 Hz, H1 offucose unit), 5.59 (d, J =8.42 Hz, H1 of glucosamine unit). ¹³ C NMR (69MHz, CDCl₃) δ 81.9, 93.3, 101.2 (anomeric carbon atoms.

Similarly, the resin 10 be can be coupled to a galactose unit, 14, toovide resin 15 (β-glycosidic linkage), as follows: Compound 10 (184.0mg, 0.414 meq./g) is added to a solution of sulfoxide 14 (208.0 mg,0.325 mmol) and 2,6, di-tert-butylpyridine (204.3 mg, 0.975 mmol) in CH₂Cl₂ (7 mL). The suspension is cooled to -60° C. and, after 10 mins, asolution of triflic anhydride (31.7 μL, 0.1883 mmol, 0.5 eq.) in CH₁ Cl₂(5 mL) is added dropwise over a period of 20 mins. Ten minutes after theaddition is complete, the reaction is slowly warmed to -30° C., andafter 30 mins, quenched by the addition of saturated aqueous sodiumbicarbonate. The resin is isolated by filtration, washed with methanol(2×10 mL), water (2×10 mL), methanol (10 mL) and CH₂ Cl₂ (10×10 mL), anddried under vacuum overnight to give 15. obtain best results, theglycosylation procedure is repeated twice.

The product is removed from the resin using the Hg(II) reagent.Hg(OCOCF₃)₂ (325.0 mg, 0.2824 mmol) and water (a few drops) are added toa suspension of 15 (222.0 mg) in CH₂ Cl₂ (20 mL). After 5 h the mixtureis filtered through cotton. The filtrate is washed with saturated sodiumbicarbonate, dried over Na₂ SO₄, filtered, and concentrated in vacuo.The residue is purified by flash chromatography (35% EtOAC-pet. ether)to give 19.4 mg of 16 (28% yield). This product could be purifiedfurther by RP-HPLC (C-18 column, 60-95% MeCN in H₂ O over 30 mins),R_(f) 26.1 mins. ¹ H NMR (500 MHz, CDCl₃) δ 4.69 (d, J =7.69 Hz, H₁ ofgalactose residue), 5.32 (dd, J=7.32, 8.8 Hz, H₁ of glucosamineresidue). ¹³ C NMR (69 MHz, CDCl₃) δ 93.5, 98.8, 101.7 (anomericcarbons).

6.15. Preparation of p-Hydroxythiophenyl Glycoside of2-Azido-2-Deoxy-4,6-O-Benzylidene-D-Glucose and Its Use in the SolidPhase Synthesis of Lewis Blood Group Antigens

An alternative method for the stereospecific preparation of (1→3)alphaand (1→3)beta glycosidic linkages in near quantitative yield tosecondary alcohols, such as those present in the Lewis blood groupantigens (Le^(x), for example, contains a fuc-alpha-(1→3)-glcNacfragment; Le^(a), another blood group antigen, contains agal-beta-(1→3)-glcNac fragment), is described below. This alternatemethod makes use of the p-hydroxythiophenyl glycoside of2-azido-2-deoxy-4,6-O-benzylidene-D-glucose, 11, shown in FIG. 18, asthe resin bound glycosyl acceptor.

Accordingly, the compound 11 is prepared as follows:

6.15.1. Preparation of 1,3,4,6-Tetra-O-acetylmannose ##STR33##

Note: To avoid the production of 2,3,4,6-tetra-O-acetylmannose, a lowerreaction temperature (20°-25° C., c.f. 35°-40 ° C.) should be maintainedduring the addition of the sodium acetate solution.

Acetic anhydride (100 mL) is placed in a 2-neck, 500 mL round bottomedflask fitted with a thermometer. Mannose (about 100 mg) is added,followed byperchloric acid (7-8 drops from a Pasteur pipette, 70%).Mannose (total of 26.4 g, 147 mmol, 1.0 eq.) is then added in smallportions over a period of 2 h, keeping the reaction temperature in therange 40°-45° C. Once the addition is complete, the orange-brownsolution is stirred for 1 h at room temperature. This leads to formationof the pentaacetate (R_(f) 0.31, 1:1 ethyl acetate-pet. ether).

The reaction mixture is cooled to 10° C. (ice bath) and phosphorustribromide (20.9 mL, 59.5 g, 220 mmol, 1.5 eq.) is then added dropwise.Then water (11.9 mL, 659 mmol, 4.5 eq.) is added dropwise. This leads tothe formation of HBr and is quite exothermic; the reaction is cooled(ice bath) to maintain an internal temperature of 20°-25° C. Once theaddition is complete, the mixture is stirred at room temperature for 1.5h. This leads to formation of the anomeric bromide (R_(f) 0.64, 2:1ethyl acetate-pet. ether).

The reaction mixture is cooled to 5 ° C. (ice bath) and a solution ofsodium acetate trihydrate (79.8 g, 586 mmol, 4.0 eq.) in water (100 mL)also at 5 ° C. is added dropwise. The reaction is very exothermicinitially, but slows after about one quarter of the sodium acetate hasbeen added. With cooling (ice bath) the temperature is kept at 20°-25 °C. and the addition is completed in 30 mins. The mixture is then stirredat room temperature for 20 mins.

The reaction mixture is poured onto ice and extracted with chloroform(3×120 mL). The extracts are combined and washed with ice-water (300mL), saturated aqueous NaHCO₃ containing ice (300 mL) and ice wateragain (300 mL). The yellow-orange organic solution is dried over MgSO₄,filtered and concentrated in vacuo. Diethyl ether (200 mL) is added andwithin a few minutes the desired product crystallizes out of solution.The flask is left in the freezer overnight and the product is collectedby filtration and is washed with a little cold ether, to give1,3,4,6-tetra-O-acetylmannose as a colorless crystalline solid (9.9 g,19% yield).

¹ H (270 MHz, CDCl₃) δ 2.06 (s, 3H), 2.09 (s, 3H), 2.12 (s, 3H) 2.18(s,3H), 2.61 (br.s, OH), 3.79 (ddd, J=2.3, 5.0, 9.9 Hz, H5), 4.13 (dd,J=2.3, 12.2 Hz, H6), 4.20 (br.s, H2), 4.30 (dd, J=5.0, 12.2 Hz, H6),5.04 (dd, J=3.0, 9.6 Hz, H3), 5.39 (app. t, J=9.7 Hz, H4), 5.80 (d, J=lHz, H1). ¹³ C NMR (69 MHz, CDCl₃) δ 20.5, 20.6, 61.9, 65.1, 68.3, 72.7,72.9, 91.6, 168.5, 169.5, 170.1, 170.7.

For further details, see, Deferrari, J. O. et al. Carbohydr. Res., 1967,4, 432 (1967); Deferrari, J. O. et al. Methods in CarbohydrateChemistry, 1972, 6, 365; Bovin, N. V. et al. Iz. Akad. Nauk SSSR Ser.Khim., 1981, 1638; and Kovac, P. Carbohydr. Res., 1986 153, 168.

6.15.2. Preparation of1,3,4,6-Tetra-O-acetyl-tri-fluorosulfonyl-β-D-mannopyranose ##STR34##

1,3,4,6-Tetra-O-acetylmannose (10.0 g, 28.7 mmol, 1.0 eq.) and pyridine(5.8 mL, 5.7 g, 71.8 mmol, 2.5 eq.) are dissolved in CH₂ Cl₂ (250 mL)and cooled to -25° C. (ethylene glycol-dry ice bath). Triflic anhydride(9.7 mL, 16.2 g, 57.4 mmol, 2.0 eq.) is added dropwise. Once theaddition is complete, the mixture is stirred at -25° C. for 45 mins,then diluted with CH₂ Cl₂ (250 mL) and washed with water (500 mL),saturated aqueous NaHCO₃ (500 mL) and water again (500 mL). The organicsolution is dried over MgSO₄, filtered and concentrated in vacuo. Thetriflate can be chromatographed (2:1 pet. ether-EtOAc) but is useddirectly.

¹ H NMR (270 MHz, CDCl₃) δ 2.08 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H),2.16 (s, 3H), 3.81-3.87 (m, H₅), 4.15-4.28 (m, 2H, H6), 5.15-5.34 (m,H2, H3 and H4), 5.92 (s, H1 - coupling constant too small to bedetected). ¹³ C NMR (69 MHz, CDCl₃) δ 20.3, 20.4, 20.5, 61.6 64.5, 69.5,73.2, 81.4, 88.9, 118.3 (d, J_(CF) =319 Hz), 167.9, 169.0, 169.8, 170.5.

For further details, see, Hamacher, K. Carbohydr. Res., 1984, 128, 291;and Pavliak, V. and Kovac, P. Carbohydr. Res., 1991, 210, 333.

6.15.3. Preparation of2-Amido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose ##STR35##

Sodium azide (3.7 g, 57.4 mmol, 2.0 eq.) is added to a solution oftriflate (13.8 g, 28.7 mmol, 1.0 eq.) in DMF (75 mL). The solution iswarmed to 40° C. under argon and stirred for 2 h. The mixture is cooled,diluted with CH₂ Cl₂ (500 mL), washed with water (500 mL) and brine (500mL), dried over MgSO₄, filtered and concentrated in vacuo. The residueis purified by flash chromatography, eluting with 2:1 pet. ether-EtOActo give azide (9.6g, 90% yield for 2 steps).

¹ H (270 MHz, CDCl₃) δ 2.03 (s, 3H), 2.08 (s, 3H), 2.10 (s, 3H), 2.20(s, 3H), 3.68 (t, J=9 Hz, H2), 3.83 (dq. J=2, 7.6 Hz, H5), 4.09 (d,J=12.5 Hz, H3), 4.31 (dd, J=4.3, 12.5 Hz, H4), 5.07 (p, J =9 Hz, 2×H6),5.57 (d, J=8.6 Hz, H1). ¹³ C HMR (69 MHz, CDCl₃) δ 20.3, 20.4, 20.5,20.6, 61.2, 62.4, 67.6, 72.4, 92.3, 168.3, 169.4, 169.5, 170.3.

For further details, see, Pavliak, V. and Kovac, P., supra; and Bovin,N. V. et al., Carbohydr. Res., 1981, 98, 25.

6.15.4. Preparation of 4-Hydroxylthiophenyl2-azido-2-deoxy-3,4,6-tri-O-acetyl-gluco!pyranoside ##STR36##

BF₃.OEt₂ (10.4 mL, 12.0 g, 84.6 mmol, 15 eq.) is added dropwise to asolution of the anomeric acetate (2.1 g, 5.64 mmol, 1.0 eq.) and4-hydroxythiophenol (1.98 g, 14.1 mmol, 2.5 eq.) in CH₂ Cl₂ (85 mL) atroom temperature under argon. The orange solution is then heated atreflux for 2 h, cooled, quenched by the dropwise addition of water (5mL). The mixture is diluted with CH₂ Cl₂ (200 mL) and washed with water(200 mL), then brine (200 mL). The organic layer is dried over MgSO₄,filtered and concentrated in vacuo. Flash chromatography, eluting with2:1 pet. ether-EtOAc, gives an initial mixture comprising startingmaterial contaminated with disulfide and, subsequently, pure product, asa 2:1 (α:β) mixture of anomers (1.27 g, 51%). The initial mixture can beredissolved in CH₂ Cl₂ (10 mL), along with 4-hydroxythiophenol (1.0 g)and BF₃.OEt₂ (5 mL). The resulting mixture is heated at reflux for 2 h,followed by work-up (as described above) to give a further 0.40 g ofproduct (total yield 67%).

Data for α-anomer (major): ¹ H NMR (270 MHz, CDCl₃) δ 2.06 (s, 6H), 2.10(s, 3H), 4.02-4.38 (m, 3H), 4.60-4.65 (m, 1H), 5.03 (app. t, J=9 Hz,1H), 5.23 (s, ArOH), 5.32 (app. t, J=10 Hz, 1H), 5.46 (d, J=5.3 Hz, H1),6.80 (d, J=8 Hz, 2H), 7.39 (d, J=8 Hz, 2H). ¹³ C NMR (67.5 MHz, CDCl₃) δ20.5, 20.6, 20.7, 61.5, 62.1, 68.3, 72.0, 87.4, 116.3, 122.3, 135.5,156.5, 169.9, 170.0, 170.8. Mass spectrum: m/z 439.1042 M⁺ calcd for C₁₈H₂₁ N₃ O₈ S: 439.1049!

6.15.5. Preparation of Benzylidene, 11 ##STR37##

Sodium methoxide (370 mg, 6.8 mmol, 3 eq.) is added to a solution of thetriacetate (1.0 g, 2.3mmol, 1 eq.) in methanol (100 mL). The solution isstirred at room temperature for 18 h to give the tetraol (R_(f) 0.17,9:1 CH₂ Cl₂ -MeOH). Amberlite IR-120 (plus) resin is added and thesuspension stirred for 30 mins, then filtered, washing the resin wellwith methanol. The filtrate is concentrated in vacuo, azeotroping withtoluene.

The tetraol is dissolved in THF (30 mL) and benzylidene dimethylacetal(1.0 mL, 1.0 g, 6.8 mmol, 3 eq.) and camphorsulfonic acid (106 mg, 0.46mmol, 0.2 eq.) added. The solution is stirred at 60° C. for 7 h, and afurther 10 h at room temperature. The mixture is concentrated and theproduct (as a mixture of anomers) is purified by flash chromatography,eluting with 4:1 pet. ether-EtOAc to give a 2:1 (α:β) mixture ofanomers. These anomers can be separated, if desired, under theseconditions. Typically, however, a mixture is used for attachment to theresin.

β-anomer: ¹ H NMR (270 MHz, acetone-d₆) δ 3.24 (dd, J=9.0, 10.0 Hz, 1H),3.38-3.49 (m, 2H, H6), 3.58-3.83 (m, 3H), 4.23 (dd, J=4.6, 10.2 Hz, 1H),4.52 (d, J=10.2 Hz, 1H), 5.26 (d, J=4.6 Hz, 1H), 5.54 (s, 1H), 6.89 (d,J=8.6 Hz, 2H), 7.32-7.47 (m, 7H), 8.75 (s, H). ¹³ C NMR (67.5 MHz,acetone-d₆) δ 66.5, 68.7, 70.8, 74.4, 81.2, 86.8, 101.9, 117.0, 120.2,127.1, 128.6, 129.4, 137.1, 13.86, 159.0.

α-anomer: ¹ H NMR (270 MHz, acetone-d₆) δ 3.60 (app. t, J=9 Hz, 1H),3.73 (app.t, J=10.2 Hz, 1H), 3.97 (m, 2H), 4.14 (dd, J=5.0, 10.2 Hz,1H), 4.33 (dd, J=5.0, 10.0 Hz, 1H), 5.37 (d, J=3.3 Hz, 1H), 5.45 (d, J=5Hz, 1H), 5.58 (s, 1H, CHPh), 6.87 (d, J=8.6 Hz, 2H), 7.34-7.53 (m, 7H),8.67 (s, 1H, ArOH). ¹³ C NMR (67.5 MHz acetone-d₆) δ 63.4, 64.0, 67.7,70.2, 31.5, 88.4, 101.3, 116.0, 121.7, 126.2, 127.7, 127.9, 128.6,135.5, 137.6, 157.6. spectrum: m/z 401.1021 M⁺ calcd for C₁₉ H₁₉ N₃ O₅S: 401.!

The compound 11 is then attached to the Merrifield's resin as the solidsupport (1% cross-linked chloromethylated styrene/divinylbenzenecopolymer, 200-400 mesh; see, e.g., Merrified, R. B. J. Am. Chem. Soc.,1963, 85, 2149) via the cesium salt of the phenol (cesium carbonate,methanol, room temperature, 3 h). The cesium salt is allowed to reactwith the chloromethylated resin in N-methylpyrrolidinone solvent at 55°C. for 24 h. The resin bound glycoside 12 is thus obtained with anestimated loading of 0.40-0.55 mmol/gram resin.

In particular, the following conditions can be followed:

6.15.6. Preparation of Resin-Bound Substance, 12 ##STR38##

Cesium carbonate (127.3 mg, 0.391 mmol, 1 eq.) is added to a solution of11 (312.0 mg, 0.781 mmol, 2 eq.) in methanol (10 mL) and stirred at roomtemperature under argon for 3 h. The solution is then concentrated invacuo, azeotroping with toluene. The yellow salt is dissolved withN-methyl-pyrrolidinone (15 mL) and Merrifield resin (391 mg, 1 mmolCl/g, 1 eq.) is added. The suspension is shaken mechanically at 55° C.under argon for 24 h. The sugar-derivatized resin is isolated byfiltration, washed with methanol (50 mL), water (50 mL), methanol (50mL) and CH₂ Cl₂ (100 mL), and dried under vacuum overnight to give 469mg of resin 12. The level of loading is estimated to be 0.46 mmol/g(based on mass gain).

IR(KBr) 2108 υ(N₃)!, 3421 br,υ(OH)!cm⁻¹.

To make the (1→3)alpha-linked disaccharide, 15, of FIG. 18, a solutionof sulfoxide 13 (145.1 mg, 0.267 mmol, 4.0 eq.) and2,6-di-tert-butyl-4-methylpyridine (164.7 mg, 0.802 mmol, 12.0 eq.) inCH₂ Cl₂ (7 mL) is added to resin 12 (145.3 mg, 0.46 mmol/g, ˜0.067 mmol,1 eq.) in a reactor vessel under argon. The suspension is cooled to -60°C. and, after 10 mins, a solution of triflic anhydride (23 μL, 0.391mmol, 2 eq.) in CH₂ Cl₂ (5 mL) is added dropwise over a period of 20mins. Twenty minutes after the addition is complete, the reaction iswarmed to -30° C. and, after 45 mins at -30° C., quenched by theaddition of saturated aqueous sodium bicarbonate (2 mL). The resin isisolated by filtration, washed with methanol (50 mL), water (50 mL),methanol (50 mL) and CH₂ Cl₂ (100 mL), and dried under vacuum overnightto give 14. The glycosylation is repeated, if desired, and the vessel issubsequently drained.

IR(KBr disc)2108 υ(N₃)!cm⁻¹.

6.15.7. Detachment From The Resin ##STR39##

Hg(OCOCF₃)₂ (114.3 mg, 0.268 mmol, 4 eq.) and water (a few drops) areadded to a suspension of resin 14 (205 mg, -0.06 mmol) in CH₂ Cl₂ (20mL). After 5 h the mixture is filtered through cotton, washing the resinwell with CH₂ Cl₂ (50 mL). The filtrate is washed with saturated sodiumbicarbonate (50 mL), dried over MgSO₄, filtered, and concentrated invacuo. The residue is purified by flash chromatography, eluting with 2:1pet. ether-EtOAc to give 31.7 mg of 15 (67% yield) as a 2:1 mixture ofalpha-linked lactol stereoisomers. R_(f) 0.30 (2:1 pet. ether-EtOc).

¹ H NMR (500 MHz, CDCl₃) δ 0.71 (app.t, J=7 Hz, H₆ Fuc), 5.29 (d, J=5.5Hz, H1_(ax) N₃ -Glu), 5.38 (t, J=3.3 Hz, H1_(eq) N₃ -Glu), 5.40 (d,J=3.7 Hz, H1 Fuc), 5.45, 5.47 (CPh). ¹³ C NMR (67.5 MHz, acetone-d₆) δ16.6, 16.7 (C6 Fuc); 93.6, 97.9 (C1 N₃ -Glu); 98.1, 98.2 (C1 Fuc);102.4, 102.5 (CHPh). High resolution mass spectrum: (M+Na)⁺ calcd forC₄₀ H₄₃ N₃ O₉ Na: 732.2397 (m/z observed: 732.2905).

The amount of product obtained represents an 89-96% yield for theglycosylation reaction, assuming that detachment from the resin proceedswith a yield 70-75% (or an overall yield of 67% over the glycosylationand detachment steps). It is important to note, that with the disclosedstrategy of attaching the glycosyl acceptor to the resin and keeping theglycosyl donor in solution, one can do repetitive couplings to increasethe yield of difficult glycosylations.

For the preparation of the (1→3)beta-linked disaccharide, 18, of FIG.18, the resin 12 is treated with perpivaloylated galactose sulfoxide 16in the same manner as described above. The desired β-linked disaccharideis isolated in 64% yield after cleavage from the resin, representing ayield of 85-91% over the glycosylation step. The stereochemical outcomeof the reaction is controlled, in this case, by neighboring groupparticipation of the pivaloyl group at C2 (see, e.g., Kunz, H.; Harreus,A. Liebigs Ann. Chem. 1982, 41).

In particular, the following procedures can be followed:

6.15.8. Preparation of the sulfoxide, 16 ##STR40##

2,3,4,6-Tetrapivoloyl-β-D-thiophenylgalactoside. Pivolyl chloride (2.10mL, 2.06 g, 17.1 mmol, 8 eq.) and N,N-dimethylaminopyridine (131.5 mg,1.08 mmol, 0.5 eq.) are added to a solution of β-D-thiophenylgalactoside(580.6 mg, 2.13 mmol, 1 eq.) in pyridine (10 mL). The solution is heatedat reflux overnight, cooled, diluted with CH₂ Cl₂ (120 mL), washed withwater (120 mL) and brine (120 mL). The organic layer is dried overMgSO₄, filtered and concentrated in vacuo. The residue is purified byflash chromatography, eluting with 10% EtOAc in pet. ether to give thesulfide, 1.09 g (84% yield). ##STR41##

Suylfoxide. m-Chloroperbenzoic acid (467 mg, 64% w/w, 1.73 mmol, 1.2eq.) is added to a solution of the sulfide (878.8 mg, 1.44 mmol, 1 eq.)in CH₂ Cl₂ (40 mL) at -30° C. After 30 mins at -30° C. under argon,dimethylsulfide (2.5 mL) is added and the mixture slowly warmed to roomtemperature. The solution is diluted CH₂ Cl₂ (100 mL), washed with water(100 mL), satd. aq. NaHCO₃ (100 mL) and water again (100 mL). Theorganic layer is dried over MgSO₄, filtered and concentred in vacuo. Theresidue is purified by flash chromatography, eluting with 20-50% EtOAcin pet. ether. The two diastereomers are separable.

First eluting diastereomer: ¹ H NMR (270 MHz, CDCl₃) δ 1.10 (s, 18H),1.11 (s, 9H), 1.46 (s, 9H), 3.93-4.07 (m, 3H), 4.46 (d, J=9.6 Hz, 1H),5.18 (dd, J=3.10 Hz, 1H), 5.38 (d, J=3 Hz, 1H), 5.56 (app. 5, J=9.9 Hz,1H), 7.52-7.55 (m, 3H), 7.64-7.68 (m, 2H). ¹³ C (67.5 MHz, CDCl₃) δ26.7, 26.9, 38.5, 38.6, 38.8, 60.1, 64.8, 65.9, 71.6, 74.8, 92.1, 127.0,128.5, 131.9, 137.1, 176.1, 176.8, 176.9, 177.5.

6.15.9. Formation of β-Glycosidic Linkage ##STR42##

A solution of sulfoxide 16 (184.5 mg, 0.295 mmol, 4 eq.) and2,6-di-tert-butyl-4-methylpyridine 181.9 mg, 0.886 mmol 12 eq.) in CH₂Cl₂ (8 mL) is added to resin 12 (160.5 mg, 0.46 mmol/g, ˜0.074 mmol, 1eq.) in a reactor vessel. The suspension is cooled to -60° C. and, after10 mins, a solution of triflic anhydride (25 μL, 41.7 mg, 0.148 mmol, 2eq.) in CH₂ Cl₂ (5 mL) is added dropwise over a period of 20 mins.Twenty minutes after the addition is complete, the reaction is warmed to-30° C. and kept at this temperature for 45 mins. The reaction isquenched by the addition of saturated aqueous sodium bicarbonate (2 mL).The resin is isolated by filtration, washed with methanol (50 mL), water(50 mL), methanol (50 mL) and CH₂ Cl₂ (100 mL), and dried under vacuum.The glycosylation procedure is repeated with the reagents in the sameproportions to give 203.05 mg of resin 17.

IR (KBr disc) 1735 υ(C═O)!, 2112 υ(N₃)!cm⁻¹.

6.15.10. Removal Of β-Linked Disaccharide From The Resin ##STR43##

Hg(OCOCF₃)₂ (126.3 mg, 0.296 mmol, 4 eq.) and water (a few drops) areadded to a suspension of resin 17 (203.5.0 mg,, ˜0.074 mmol, 1 eq.) inCH₂ Cl₂ (20 mL). After 5 h the mixture is filtered through cotton,rinsing the resin well with CH₂ Cl₂ (50 mL). The filtrate is washed withsaturated sodium bicarbonate, then brine and dried over MgSO₄, filtered,and concentrated in vacuo. The residue is purified by flashchromatography, eluting with 3:1 petroleum ether-ethyl acetate to give37.3 mg of 18 as a 3:2 mixture of beta-linked lactol stereoisomers (64%yield). R_(f) 0.33 (3:1 pet. ether-EtOAc).

¹ H NMR (270 MHz, CDCl₃) δ 1.10, 1.11, 1.12, 1.13, 1.19, 1.23 CH₃ groupsof Piv!, 4.85 (d, J=7.9 Hz, H1 Gal), 5.24 (d, J=7.9 Hz, H1 N₃ -Glumajor)), 5.28 (d, J=7.9 Hz, H1 N₃ -Glu minor), 5.54 (s, 1H, CHPh). ¹³ CNMR (69 MHz, CDCl₃) δ 27.9, 27.1 (CH₃ of Piv), 38.6, 38. 8, 38.9, 39.9(CHMe₃ of Piv); 92.5, 96.9 (C1 of N₃ -Glu); 100.4, 100.5 (C1 of Gal);101.1, 101.6 (CHPh); 176.7, 176.9, 177.4, 177.7 (C═O of Piv). Highresolution mass spectrum: (M+Na)⁺ calcd for C₃₉ H₅₇ N₃ O₁₄ Na:814.3738(m/z observed:814.3727).

Accordingly, the present disclosure establishes that the sulfoxideglycosylation reaction can be used to form both alpha and betaglycosidic linkages stereospecifically and in high yield to secondaryalcohols on an insoluble support.

6.16. Standard References

Most of the transformations mentioned above (protection: benzylation,benzylidenation, acetonation, esterification, and carbo- orsilylethentication of sugars; deprotection: debenzylation, acidichydrolysis of benzylidenes or acetonates, basic hydrolysis of esters,removal of silyl groups with fluoride or under acidic conditions) aredescribed in Binkley, R. W. Modern Carbohydrate Chemistry, MarcelDekker, Inc.: New York, 1988. Methods to convert lactols or anomericesters or anomeric esters to thiophenyl groups (to produce thiophenylglycosides) are well known. See, e.g., Ferrier et al. Carbohydr Res.1973, 27, 55; Mukaiyama et al. Chem. Lett. 1979, 487; Van CleveCarbohydr. Res. 1979, 70, 161; Hanessian et al. Carbohydr Res. 1988, 80,C17; Garegg et al. Carbohydr. Res. 1983, 116, 162; and Nicolaou et al.J. Am. Chem. Soc. 1983, 105, 2430.

From the principles established herein, it should be apparent to one ofordinary skill in the art that the ability to manipulate the reactivityof both glycosyl donors and glycosyl acceptors to control the order inwhich glycosylation takes place can be exploited to synthesize manyother oligosaccharides or glycoconjugates rapidly, efficiently and inhigh yield, under either homogeneous (in solution) or heterogeous (inthe solid phase) conditions.

What is claimed is:
 1. A compound of the formula (IV): ##STR44## inwhich each of the groups R₁, R₂, and R₃ can independently be a hydrogenlower alkyl or a hydroxyl protecting group, the group R₄ can be ahydrogen, lower alkyl, a hydroxyl protecting group, a covalent linkermoiety, and the group N* is a masked group convertible to an aminogroup.
 2. The compound of claim 1 in which the group R₁ is a hydrogenand the groups R₂ and R₃ singly or together comprise a hydroxylprotecting group.
 3. The compound of claim 2 in which the group R₁ is ahydrogen.
 4. The compound of claim 1 in which the masked group N*comprises an azido group.
 5. The compound of claim 1 in which the maskedgroup N* comprises a phthalimide group.
 6. A compound of the formula(V): ##STR45## in which the group R₁ can be a hydrogen, lower alkyl or ahydroxyl protecting group, the group R₄ can be a hydrogen, lower alkyl,a hydroxyl protecting group, a covalent linker moiety and the group N*is a masked group convertible to an amino group.
 7. The compound ofclaim 6 in which the groups R₁ and R₄ are hydrogen.
 8. The compound ofclaim 6 in which the masked group N* comprises an azido group.
 9. Asubstance of the formula (VI): ##STR46## in which each of the groups R₁,R₂, and R₃ can independently be a hydrogen, lower alkyl or a hydroxylprotecting group, the group N* is a masked group convertible to an aminogroup, the group "Resin" comprises an insoluble solid support, and thegroup X comprises a covalent linker moiety.
 10. The substance of claim 9in which said "Resin" comprises a crosslinked styrene/divinylbenzenecopolymer.
 11. The substance of claim 9 in which said "Resin" comprisesa Merrifield resin.
 12. The substance of claim 9 in which said "Resin"polystyrene.
 13. The substance of claim 9 in which said "Resin"comprises a PEG-derivatized polystyrene.
 14. The substance of claim 9 inwhich the group X comprises an ether group.
 15. The substance of claim 9in which the group X comprises an ester group.
 16. A compound which is4-hydroxythiophenyl- 2-azido-2-deoxygluco!pyranoside.
 17. The compoundof claim 16 in which one, two or three hydroxyl groups is (are)protected by a protecting group.
 18. The compound of claim 16 which isthe 3,4,6-tri-O-acetyl derivative.
 19. The compound of claim 16 which isthe 4,6-O-benzylidene derivative.
 20. The substance of claim 9, whereinsaid "Resin" swells in organic solvents.
 21. The substance of claim 9,in which the group X comprises an aliphatic group.
 22. The substance ofclaim 9, in which the group X comprises an aromatic group.
 23. Thesubstance of claim 9, in which the group X comprises an amide group.